https://orcid.org/0000-0001-6648-2960
Figures
Abstract
The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5’-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.
Citation: Inokuchi Y, Quaglia F, Hirashima A, Yamamoto Y, Kaji H, Kaji A (2023) Role of ribosome recycling factor in natural termination and translational coupling as a ribosome releasing factor. PLoS ONE 18(2): e0282091. https://doi.org/10.1371/journal.pone.0282091
Editor: Hari S. Misra, Bhabha Atomic Research Centre, INDIA
Received: October 14, 2022; Accepted: February 7, 2023; Published: February 24, 2023
Copyright: © 2023 Inokuchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: No The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ribosome recycling factor was first found to be an enzyme that releases ribosomes from mRNA for a new cycle of translation. Hence, the factor was originally called “ribosome releasing factor (RRF)” [1] and the gene, frr, and the promoter position were identified near 4 minutes in the E. coli genetic map [2]. For early review see [3,4]. Later, the factor was re-named “ribosome recycling factor” [5] because its function is to recycle the used ribosomes by releasing them from mRNA and tRNA [6]. One additional function, splitting of ribosomes of the PoTC (post-termination complex) during recycling, was discovered through kinetic analysis of formation of oligopeptides [7]. The ribosome splitting activity of RRF was confirmed later by three independent laboratories [8–10]. The recycling process is an essential step in all organisms so far studied [5,11–14].
When RRF is absent, ribosomes stay at the termination codon and start translating from the next codon [15]. This is called “unscheduled translation” [16] because translation beyond the termination codon is not scheduled. This is somewhat similar to the term re-initiation but we prefer to use the term “unscheduled translation” specifically referring to translation due to failure of ribosome recycling. There is another factor, IF3, that helps dissociation of 70S ribosomes [17,18]. It is likely that in the absence of RRF, the role of IF3 [19] as the second splitting factor would become important. RRF is also suggested to function as an agent dealing with stalled ribosome [20].
RRF has a striking structural similarity to tRNA [21]. Studies suggest that RRF binds to 70S ribosomes covering the A/P site [22–26] and moves within the inter-ribosomal space to the P-site [27–29] like tRNA. The RRF reaction is dependent on GTP [30–33] just like the movement of tRNA during translocation. However, the orientation of ribosome-bound RRF is upside-down compared to that of tRNA. In a recent publication using time-resolved cryo-EM, which makes it possible to visualize short-lived intermediates, the mRNA was never released from the ribosome due to proximity of the Shine-Dalgarno (SD) sequence to the termination codon [29]. Another paper utilized a novel single-molecule fluorescence technique to visualize the interactions of molecules in ribosome recycling [34]. However, their results showed a very late and slow release of 30S ribosomal subunit from the mRNA, which may be due to the presence of a SD sequence near a very short ORF. A recent report grants an interesting view of the crystal structure of a ribosome with two tRNAs, RRF and EF-G [35]. The role of this complex in the RRF reaction remains to be elucidated.
The overlapping sequence at the border region between two adjacent ORFs in a polycistronic mRNA was first discovered at the border region of trpB and trpA [36]. It was thought to be peculiar to the case of translational coupling where a certain ratio of a pair of proteins is required for the function of two proteins. However, the complete sequencing of bacterial genomes suggests that such sequences are common in the inter-cistronic region in an operon in E. coli [37]. We define translationally coupled ORFs as a pair of ORFs where reading of the downstream ORF (d-ORF) is performed, totally or partially, by the ribosome(s) which have completed translation of the upstream ORF (u-ORF) [38]. In our preceding publication, we showed that, in RNA phage GA, the translation of the lysis protein gene (d-ORF) without an SD sequence was performed strictly by the ribosomes that translated the coat protein gene (u-ORF) [39]. The deletion and base substitution analyses revealed that there were no sequences that allowed the expression of the downstream lysis gene independently of the translation of the upstream coat gene. During this process, the ribosomes accomplish the frameshift required by the overlapping initiation and termination codons.
In this paper, we investigated functions of RRF during the natural termination process using the translationally coupled system without the SD sequence of d-ORF. Our data, both in vivo and in vitro, are consistent with the interpretation that RRF releases ribosomes from the termination codon of the junction sequence, which allows the released ribosome to bind to the nearby initiation triplet of d-ORF. In the in vivo experiments, this basic behavior of ribosomes is influenced by the sequence surrounding the termination triplet. When u-ORF was very short, d-ORF reading was not observed at all. Furthermore, in the in vitro experiments, use of genetically conjugated ribosomal subunits [40] showed that the recycling of ribosomes at the junction sequence can occur without complete splitting of the 70S ribosomes of PoTC. This indicates that our original concept of RRF as the “ribosome releasing factor” [1,41] is correct in that the major function of RRF resides in the release of ribosomes from mRNA of PoTC.
Results
Translationally coupled ORF without the SD sequence for d-ORF gives a way to study the role of RRF at the termination step
The in vivo behavior of ribosomes that just finished termination step is not well understood. However, it is known that, in translational coupling, ribosomes which are finishing the translation of u-ORF (u-ribosomes) are responsible for the translation of d-ORF without SD [39,42]. The lysis gene of RNA phage GA is exclusively read by the ribosomes that have completed the reading of the upstream coat gene. This is achieved by the unique junction sequence UAAUG present between these two ORFs. On this basis, we describe here series of experiments indicating the role of RRF on the behavior of u-ribosomes by using such translational coupling.
To investigate the role of RRF in ribosome recycling in vivo, we constructed plasmids having the cDNA of GA phage including the region between the coat and lysis genes [39]. With these plasmids, the lysis gene was replaced by the reporter lacZ gene to study d-ORF expression precisely. Furthermore, to see whether the coupling system works only with the junction sequence UAAUG, we examined the sequence AUGA as well. As shown in Fig 1, a reporter lacZ gene (d-ORF) coding for β-galactosidase was connected to the C-terminal sequence of GA phage coat gene (u-ORF) through the junction sequence UAAUG in (A) or AUGA in (B). UAA and UGA are termination triplets of u-ORF. AUG is in frame with the lacZ gene. We define the reading frame of AUG as zero frame in this paper. The plasmids express the reporter gene through translational coupling. To control the RRF activity in vivo, we used the E. coli strain LJ14, which contained a temperature sensitive mutation of RRF (tsRRF). With this strain, tsRRF is active at 31°C while it is inactive at 37°C [16].
Diagrams of plasmids (A) and (B) are shown. Initiation codon and termination codons are shown in capital letter and highlighted for clarity. AUG is the initiation codon for upstream open reading frame (u-ORF) and AUG is that for downstream open reading frame (d-ORF). The initiation codon for d-ORF is overlapped with the termination codon for u-ORF ((A) UAA and (B) UGA). u-ORF and d-ORF are boxed. Nucleotide sequence 5’-AGGA-3’ shown in italic represents a Shine-Dalgarno (SD) sequence of u-ORF. LacZ represents the reporter lacZ(Δ27) gene for β-galactosidase. n in the bracket represents a nucleotide and numbers next to the bracket represent the number of nucleotides in the bracket. The nucleotide sequence labelled (n)88 is referred to [39,43] for details.
Fig 2 shows that our translational coupling system described above confirmed the following characteristics of the system by the coat-lysis genes pair. First, all ribosomes that translate d-ORF are derived from u-ribosomes. When u-ORF was lost (panels (b) and (d)), no d-ORF reading was observed with the junction sequences UAAUG (compare (b) with (a)) and AUGA (compare (d) with (c)). Free ribosomes are excluded from engaging in translation of d-ORF without SD. Second, u-ribosomes are released at the termination codon of u-ORF and re-bind to the initiation codon of d-ORF (frameshift). This is because the reading frame of u-ORF differs from that of d-ORF. Panel (e) indicates that triplet aUA (coding for isoleucine but not for methionine) did not function as the initiation codon for d-ORF (compare panel (e) with (c)). Third, u-ribosomes move both directions at the termination codon. As shown in panels (a) and (c), the released u-ribosomes bound to AUG situated downstream of the termination codon of u-ORF (panel (a)) as well as AUG situated upstream of that (c).
E. coli LJ14 cells harboring plasmids were grown at 28°C to a cell density of ~0.13 as described in Experimental procedures and IPTG (final 2 mM) was added to the culture for induction of the reporter lacZ(Δ27) gene. The culture was incubated for another two hours. At the indicated times after the addition of IPTG, an aliquot was taken and the β-galactosidase activity was measured. The junction sequences and presence (+) or absence (−) of u-ORF are shown above each panel. The nucleotide sequences around the junction sequence are shown at the bottom. Rep represents the reporter lacZ(Δ27) gene. Dotted lines (……) in (b) through (e) represent the same nucleotide sequences with that of (a) in the corresponding regions. Panels (a) and (c) represent the β-galactosidase synthesis through translational coupling. (b) and (d) are identical to (a) and (c), respectively, with the exception that AUG of u-ORF was changed to auu. (e) is identical with (c) with the exception that (e) has aUAA instead of AUGA. The reporter lacZ gene is in frame with AUG of UAAUG, AUGA and aUA of aUAA.
In vivo evidence that RRF releases all ribosomes from the termination triplet
We have demonstrated that RRF releases mRNA from PoTC during ribosome recycling in vitro [1,24,41]. This predicts that, in our in vivo translational coupling system, all u-ribosomes would be released by RRF from UAA of the junction sequence UAAUG. Some of the released u-ribosomes would re-bind to AUG of UAAUG, followed by d-ORF (reporter gene) reading. To examine this prediction, we constructed three kinds of plasmids in which the reporter lacZ gene was connected to the junction sequence in three reading frames to observe all frames of translation. We then compared the amounts of ribosomes engaging in downstream reading (β-galactosidase activity) in the presence and absence of RRF. Our prediction that RRF releases all ribosomes from PoTC in vivo was fulfilled as shown in Fig 3 and Table 1.
Numbers in square brackets shown above each panel represent the reading frame of d-ORF counting from AUG or Aua of UAAUG or UAAua, respectively, shown on the right side of panels. Panel (a) (a control plasmid) has no junction sequence and represents the u-ORF reading. The scale of the β-galactosidase activity in panel (a) is four times larger than that of (b) through (g). Panel (b) has the junction sequence UAAUG and represents the β-galactosidase synthesis through the coupling mechanism. The reporter lacZ(Δ27) gene of (b) is in zero frame (in frame with AUG of UAAUG). Panels (c) and (d) represent the cases in which the reporter gene is in +1 (c) and +2 (d) frame, respectively, counting from AUG of UAAUG. Panels (e), (f) and (g) have the sequence UAAua instead of UAAUG. Dotted lines (……) represent the same nucleotide sequences with that of (b) in the corresponding regions.
In this figure, with the junction sequence UAAUG, d-ORF reading (β-galactosidase activity) was observed in 0 frame (panel (b)) but not in +1 (c) and +2 (d) frames in the presence of RRF (open circles). This is because u-ribosomes were released at UAA and bound back specifically to AUG of UAAUG. On the contrary, in the absence of RRF activity (closed circles), d-ORF reading was also observed in +1 (c) and +2 (d) frames. This indicates that u-ribosomes remained on the mRNA and re-initiated translation in all three frames. Furthermore, when AUG of UAAUG was changed to Aua (Fig 3 panels (e), (f) and (g)), d-ORF reading was not observed in any frame in the presence of RRF (open circles), indicating that u-ribosomes were released from RNA. However, in the absence of RRF activity (closed circles), d-ORF reading was observed in every reading frame, indicating that u-ribosomes remained on the mRNA.
To estimate how much of u-ribosomes would stay on the mRNA, percentages of the u-ribosomes engaged in d-ORF reading were calculated from Fig 3 and summarized in Table 1. It is clear from this table (column 7, All frames) that more u-ribosomes read downstream in the absence of RRF activity than in the presence of that (UAAUG: 54.3% vs 25.5%, UAAua: 46.1% vs 2.2%). However, the total amount of d-ORF readings (54.3% and 46.1%) were less than 100% of u-ORF reading. This is because, at the non-permissive temperature for tsRRF, the ribosomes that completed ORF reading often slide toward the 3’ direction without translation and resume translation at random [16]. Protein products made by ribosomes that resume translation within the reporter lacZ gene would not have the enzyme activity and will not be counted in our assay. It is therefore assumed that, in the absence of RRF, the actual number of mRNA-bound u-ribosomes engaged in downstream reading would be higher than the value shown in Table 1. From these considerations, we conclude that most, if not all, ribosomes remained on mRNA in the absence of RRF activity.
The effects of temperature on wild-type RRF regarding the reporter gene expression have been reported [16]. In that paper, we have shown that, with normal ORF, downstream reading was not observed when the culture temperature was shifted from 31 to 39°C. Furthermore, LJ14 cells harboring plasmid pPEN907 carrying wild-type frr showed wild-type phenotype at the permissive and non-permissive culture temperature (Fig 4 panel B of [16]). These results indicate that, with wild-type RRF, the change of culture temperature does not cause the change of the amount of downstream reading in our system.
LJ14 cells grown at 28°C were divided into 5 parts and further incubated at various temperatures. Numbers in square brackets shown on the right side of panels represent the reading frame of d-ORF counting from AUG of the junction sequence UAAUG. In panels (a)—(e), AUG of UAAUG is in frame with the reporter lacZ(Δ27) gene. In (f)—(j) and (k)—(o), AUG of UAAUG is out of frame with the reporter gene. The nucleotide sequences labelled (n)n are 5’-gguctcaaagcaaaacacaaggaaaacctcgcgaaauacgua-3’ (panels (a)–(e)), 5’-gguctcaaagcaaaacacaaggaaaacctcgcgauacgua-3’ ((f)–(j)), and 5’-gguctcaaagcaaaacacaaggaaaacctcgcgaauacgua-3’ ((k)–(o)). At 39°C, the temperature is high enough to inactivate RRF and cause thermal frameshift, which is shown by reading at all three frames (panels (e), (j) and (o)).
At the non-permissive temperature of tsRRF, ribosomes remaining at the termination codon undergo frameshift to all frames
Structural studies [24,28,29,44] showed that the natural substrate of RRF does not have a peptidyl group which functions to stabilize the elongating ribosomes. Based on this consideration, we postulated that the ribosome, by the inactivation of tsRRF, would stay at the UAA codon without stabilization by peptidyl group. Because of the high culture temperature (39°C) used to inactivate tsRRF, these ribosomes would then start vibrating (thermal vibration) at UAA, and would be frameshifted to all frames. This prediction was fulfilled as shown in Fig 3 (closed circles).
In this figure, we show that, at 39°C where RRF activity was absent, all frames were read (panel (b) to (g)). This indicates that ribosomes which finished u-ORF reading at the UAA frameshifted to all frames. We should recall that, without RRF, ribosomes read downstream only in frame with the termination codon in vitro [15]. If ribosomes behave similarly in vivo as in vitro, when we inactivate tsRRF, we would see reading of the downstream reporter gene only in frame with UAA. In other words, ribosomes would read +1 frame only (panels (c) and (f)), closed circles). On the contrary, we saw the downstream readings in all frames. This is because, due to the high culture temperature, temperature-dependent frameshift to all frames took place for ribosomes that remained on the UAA.
Both inactivation of RRF and high culture temperature are necessary for frameshift to all frames
We postulate that there are two elements influencing frameshift; high culture-temperature and loss of RRF activity. To sort out the temperature effect and the effect of the loss of RRF activity on frameshift at the junction sequence UAAUG, we tested the system at various temperatures for frameshift to all frames or to zero frame only (from UAA to AUG). The results are shown in Fig 4.
As shown in panels (c), (h), and (m), only frameshift to zero frame (AUG) occurred at 35°C by ribosomes released by remaining RRF activity. At 37°C, where tsRRF is inactivated [16], preferential reading of AUG began to wane (panel (n)) because temperature-dependent vibration of ribosomes occurred. However, panels (d) and (i) show that even at 37°C the remaining RRF activity is sufficient to release ribosomes from UAA to bind back to AUG. Since most of the ribosomes read the frame starting from AUG, temperature-dependent frameshift to all frames was not observed. At 39°C, frameshift to all three frames was observed, indicating that RRF activity was lost resulting in loss of AUG-specific reading. Frameshift to all frames due to high culture temperature begins here. Therefore, we conclude that both complete inactivation of RRF and a high culture temperature are required for temperature-dependent frameshifting. In fact, in vitro experiments described in the later section of this paper show that removal of RRF alone does not induce frameshift to all frames. The above conclusion is also consistent with the fact that no published paper has shown any frameshift at high temperature using wild-type E. coli.
The distance between AUG and UAA in the junction sequence influences d-ORF reading only in the presence of RRF: Further support for RRF-dependent release of ribosomes from PoTC
Up to this point, we presented the cases where termination codon of u-ORF and initiation codon of d-ORF overlap (for example, UAAUG). In the experiments shown in Fig 5, we studied the effect of the distance between UAA and AUG on d-ORF reading. We reasoned that ribosomes can start reading from any position on mRNA as long as it remains attached to the mRNA. On the other hand, ribosomes once released from the mRNA have to seek AUG to re-start the translation. Therefore, one would expect that the distance would make difference on the downstream reading only in the presence of RRF. The amount of d-ORF reading would decrease as the distance between UAA and AUG increases. This prediction was fulfilled as described below.
Nucleotide sequence and the distance (D and d) between AUG and UAA at the junction regions are indicated above each panel. AUG is the initiation codon of d-ORF and in frame with the reporter lacZ(Δ27) gene. UAA is the termination codon of u-ORF. D represents distance in nucleotides when AUG is upstream of UAA while d represents distance when AUG is downstream of UAA. The minus sign (−) in panel (d) indicates that UAA and AUG are overlapped by one nucleotide (d = −1). Cases where AUG is located upstream of UAA with distance 3 and 0 nucleotides (D = 3 and 0) are not shown because AUG is in frame with downstream UAA.
As shown in panels (a) through (c) of Fig 5, when AUG was situated upstream of UAA, there was a distinct effect of the distance (shown as D) between AUG and UAA on d-ORF reading (open circles). The β-galactosidase activity in (a) (D was 4 nucleotides) was about 20% of that in (c) (D was one nucleotide) at 2 hrs. after IPTG addition. The amount of d-ORF reading decreased as the distance between AUG and UAA increased. These are cases where ribosomes must move backwards from 3’ to 5’ direction (against the natural direction of ribosome movement) to bind to AUG.
In contrast, in panels (d) through (g) (open circles), when the released ribosomes must bind to AUG situated downstream from UAA, the expression of the reporter gene was approximately equal in all cases regardless of the distance (d equals -1, 0, 1 and 4 nucleotides). We interpret that the moment of inertia of the ribosomes moving toward the 3’ direction masks the effect of distance when the distance is only a few nucleotides.
What is the effect of the distance and direction of ribosome movement in the absence of RRF? In these cases, the ribosomes which completed u-ORF reading were allowed to move toward the 3’ direction on the mRNA and resume translation from random places [16]. Therefore, for the ribosomes remaining at UAA, the distance between AUG and UAA and the relative position of these triplets would have no effect on d-ORF reading. This was true as shown in panels (a) through (g) (closed circles). We conclude from Fig 5 that ribosomes were released at UAA from mRNA of PoTC by RRF and rebound to AUG of the junction sequence.
The SD sequence near the junction region attracts mRNA-bound ribosomes as well as released ribosomes after termination
In most of studies carried out in vitro to investigate the RRF function, mRNA contains the SD sequence near the termination triplet [8,29,31,34]. This is because the termination complex used was prepared with short mRNA. This results in the termination complex near SD. Since there is no SD near the natural termination triplet, we decided to elucidate the effect of SD on the behavior of ribosomes in the translational coupling between a short u-ORF with SD and d-ORF without SD. The ribosomes released from u-ORF would be attracted by the SD to re-read mostly the short u-ORF. Therefore, one would expect no reading of the d-ORF. In other words, the size of u-ORF is important for determining the amount of d-ORF reading by the ribosomes that have finished reading the u-ORF. A short u-ORF would abolish the expression of the reporter gene connected to the d-ORF. On the other hand, in the absence of RRF activity, u-ribosomes would be less influenced by SD because they are bound to mRNA. These expectations were fulfilled as shown in Fig 6.
Numbers of codons in u-ORF are shown above each panel. SD represents the SD sequence (5’-AGGA-3’). AUG of the junction sequence UAAUG is in frame with the reporter lacZ(Δ27) gene. n in the bracket represents a nucleotide and numbers next to the bracket show the number of nucleotides in the bracket. The nucleotide sequences around the junction sequence are shown at the bottom. Dotted lines (……) in (b) through (e) represent the same nucleotide sequences with that of (a) in the corresponding regions. Panel (a) has no junction sequence and represents the u-ORF reading. The nucleotide sequences labelled (n)n are 5’-aca-3’, 5’-accauc-3’, 5’-accaugauu-3’, 5’-accaugauuacc-3’, and 5’-accaugauuacgaau-3’ for panel (b), (c), (d), (e) and (f), respectively. Dotted lines (……) represent the same nucleotide sequences with that of panel (a) in the corresponding regions.
In Figs 6 and 7, we designate the initiation signal of u-ORF as AUG and that of d-ORF as AUG. The termination codons of u-ORF are UAA or UGA, and we assume that UAA and UGA work similarly in all cases. As shown in Fig 6 panels (b), (c) and (d) (open circles), when u-ORF was shortened to three to five codons, there was no reporter gene expression. This suggests that all the ribosomes released by RRF from the termination triplet UAA of u-ORF were attracted by SD of the short u-ORF and re-read from the AUG to translate u-ORF but not d-ORF. On the other hand, when u-ORF was more than six codons, d-ORF was read ((e) and (f), open circles). This is because ribosomes released by RRF from UAA rebound to nearby AUG. These data suggest that, when u-ORF is more than six codons, the pulling force of the SD is weak enough for released ribosomes to be attracted by SD.
Numbers of codons in u-ORF are shown above each panel. SD represents the SD sequence (5’-AGGA-3’). UAA and UGA are the termination codons of u-ORF. AUG is the initiation codon of d-ORF and in frame with the reporter lacZ(Δ27) gene. Dotted lines (……) represent the same nucleotide sequences with that of the plasmid of panel (a) in the corresponding regions. Panel (a) has no junction sequence representing the u-ORF reading. In panel (b), AUG is downstream of UAA. In (c), AUG is upstream of UGA. In (d), termination codon UGA of u-ORF is 94 nucleotides far from AUG and exists within the reporter gene.
In the absence of RRF (closed circles), the size of u-ORF also has a similar effect on d-ORF reading. With 4, 5, 6 and 7 codons of u-ORF, the d-ORF was read (Fig 6 panels (c), (d), (e) and (f)). When u-ORF was shortened to three codons, d-ORF was not read (panel (b)). These data demonstrate that SD also influences the behavior of ribosomes remaining on the mRNA after termination. It should be mentioned that the expression of d-ORF was higher in 5 codon u-ORF (d) than in 6 codon u-ORF (e). This is contradicting with our expectations. We discuss this difference in Discussion section. The effect of SD was much stronger on ribosomes released by RRF than on ribosomes that remained on mRNA. This is because, in the presence of RRF (open circles), the effect was observable with 5 codon u-ORF (panel (d)). On the other hand, ribosomes remaining on the mRNA due to the loss of RRF showed the effect of SD with 3 codon u-ORF ((b), closed circles).
Relative positions of the start and stop codons of the junction sequence with respect to SD determine the amount of d-ORF reading
In addition to the size of u-ORF, the relative positions of the start and stop codons of the junction sequence with respect to the SD sequence of short u-ORF should also determine the effect of SD on ribosomal behavior. We call this the directional effect of SD. As described in the preceding chapter, RRF-released ribosomes should be attracted toward upstream by the SD. Therefore, if the initiation codon of the d-ORF is upstream of the termination codon of the u-ORF, SD will help the released ribosomes move toward the initiation codon of the d-ORF and bind to it resulting in d-ORF reading. We predict that AUG of d-ORF situated upstream of UAA or UGA of u-ORF would stimulate d-ORF reading.
This expectation was met as shown in Fig 7. In panel (c) (junction sequence AUGA), where the initiation codon AUG was on the SD side of the termination codon UGA, there was a significant amount of d-ORF reading (open circles). This is because released ribosomes bind to the initiation triplet AUG on the way toward the SD. On the contrary (panel (b) (UAAUG)), there was no d-ORF reading when the initiation codon AUG was on the opposite side of SD of the termination codon UAA. The significant d-ORF reading (c) was not due to mRNA-free ribosomes attracted by SD, because when the termination codon UGA was moved 94 nucleotides downstream away from the initiation codon AUG, no d-ORF reading was observed (d).
In the absence of RRF (Fig 7, closed circles), we also observed the directional effect of SD on d-ORF reading by ribosomes that remained on the mRNA. As shown in panels (b) (junction sequence UAAUG) and (c) (AUGA), downstream reading was observed when AUG was on the side of SD (c) but not when AUG was on the opposite side of SD (b). These data support the idea that a nearby SD attracts RRF-released ribosomes as well as mRNA-bound ribosomes.
In vitro evidence suggests that RRF acts as a ribosome releasing factor in translational coupling without SD of d-ORF
In this paper, we emphasize the importance of RRF action on the release of ribosomes from the PoTC. As a model, we chose translational coupling because the u-ribosomes must be used for reading from the initiation triplet of d-ORF without the SD sequence. This is accomplished by releasing u-ribosomes from the termination codon followed by rebinding of the ribosomes to the nearby initiation triplet of d-ORF. During this process, frameshift may occur. We investigated the essential nature of the RRF action in translational coupling as a frameshift causing agent in the in vitro system as described in this section.
Here, we studied translational coupling through in vitro experiments using 027mRNA [45] having the junction sequence UAAUG without an SD sequence nearby (Fig 8). The u-ORF (shown in italic) contains triplets UUU and UUC coding for phenylalanine. Therefore, using this mRNA with the PURE system [46], we expect phenylalanine incorporation into polypeptides as indication of upstream reading. For downstream reading, there are two possibilities. First, in the presence of RRF, ribosomes are released from UAA by RRF and bind to AUG of UAAUG on the same mRNA. The ribosome will then read triplets CGU (arginine) followed by CUG (leucine) because they are in frame with AUG of the junction sequence. Second, in the absence of RRF, ribosomes will remain on UAA and will start reading from the next codon, UGC (cysteine), followed by GUC (valine) as observed by Ryoji et al [15]. Therefore, for downstream reading, we expect leucine (0 frame, the AUG frame) or valine (+1 frame) incorporation in the presence or absence of RRF, respectively. These expectations were met as shown in Figs 9–11.
Two ways of downstream reading that depend on the presence or absence of RRF are shown. u-ORF is in italic. SD and the junction sequence are in bold. Amino acid residues corresponding to the triplets of the reading frames are shown in single capital letters.
u-ORF reading was measured by incorporation of [3H]-Phenylalanine coded for by UUU and UUC codons into hot TCA insoluble materials. Reaction mixtures were as described in Materials and Methods. The concentration of Mg2+ was 4 mM.
A RRF stimulates downstream reading in frame with AUG. Downstream reading was measured by incorporation of [14C]-Leucine coded for by CUG and CUA codons in frame with AUG. Reaction mixture was identical to that of Fig 9 except for labeled amino acid (leucine). Mg2+ concentration was 4 mM. B At high Mg2+ concentration, downstream reading in frame with AUG is more dependent on RRF. The reaction mixture was identical to that of Fig 10A except that Mg2+ concentration was 7 mM to observe ribosome releasing activity of RRF more clearly. Note that requirement for RRF is clearer under the condition (7 mM Mg2+) where non-enzymatic release of ribosomes from mRNA is harder than that in 4 mM Mg2+.
Downstream reading in frame with UAA of the junction sequence was measured by [14C]-Valine incorporation coded for by triplet GUC which was in frame with UAA. The components of the reaction mixture were identical to that of Fig 9 except for labeled amino acid (valine).
As shown in Fig 9, phenylalanine incorporation in the presence of RRF (open circles) was much higher than in the absence of RRF (closed circles). The data support the hypothesis that RRF releases the ribosomes at UAA of UAAUG, which allow them to be used in the next cycle of u-ORF translation. In the absence of RRF, the reduced incorporation of phenylalanine is due to reduced number of recycled ribosomes.
What about the downstream reading? As shown in Fig 10A, RRF induced additional leucine incorporation (0 frame reading starting from AUG, see the sequence below the figure) (open circles). This indicates that ribosomes which have finished u-ORF translation at UAA (+1 frame) initiated downstream reading from AUG (0 frame). However, even in the absence of RRF (closed circles), comparable amounts of leucine were incorporated. This is because some of the ribosomes at UAA were released from mRNA non-enzymatically at low (4 mM) Mg2+ and rebound to AUG of UAAUG resulting in leucine incorporation. Therefore, one would expect that leucine incorporation in the absence of RRF should be reduced at higher Mg2+ concentration. This is because Mg2+ increases the affinity of RNA to ribosomes [47]. Indeed, as shown in Fig 10B, leucine incorporation in the absence of RRF (closed circles) became much less at 7 mM Mg2+ compared with that at 4 mM Mg2+ (Fig 10A). Consequently, the dependency of leucine incorporation on RRF became more distinct at high Mg2+.
In the case of plus one frame reading of the downstream region (Fig 11), labeled valine was incorporated in the absence of RRF (closed circles). In fact, the sequence of 027mRNA codes for valine only when ribosomes translate after the junction sequence in frame with UAA (+1 frame). This indicates that ribosomes remaining at the junction sequence UAAUG did not recognize the AUG as an initiation codon. In the presence of RRF (open circles), no significant amount of valine was incorporated because ribosomes were released by RRF at UAA and re-started reading from AUG of the junction sequence. In these experiments the Mg2+ concentration was 4 mM which is unfavorable for the RRF reaction due to non-enzymatic release of ribosomes at lower Mg2+. Even in this unfavorable condition, RRF exerted its effect of releasing ribosomes from mRNA resulting in decreased incorporation of valine.
Complete ribosome splitting is not necessary for the release of ribosomes from mRNA
It has been assumed that the ribosome splitting is a part of ribosome recycling because subunits have been the major player of the initiation which follows the recycling step.
However, in 2015, an E. coli strain which contains 70S ribosomes that do not split was reported [40]. This strain demonstrates that complete ribosome splitting is not essential for cell growth. In this section, we examined whether the non-dissociable ribosomes (ribo-T) function at the junction sequence, UAAUG, similarly to the normal ribosomes in the presence and absence of RRF in vitro.
It is clear from Fig 12A that, even in the presence of RRF, ribo-T was unable to dissociate into 30S and 50S subunits but converted to 65S particles [40]. On the other hand, the wild-type ribosomes were dissociated into 50S and 30S subunits in the presence of RRF. Using the in vitro translational coupling system as described above, we show that ribo-T is released from mRNA by RRF at UAA of UAAUG as shown in Fig 12B.
A ribo-T is not split while wild-type 70S ribosomes are split into 30S and 50S subunits in the presence of RRF. Upper figures show wild-type ribosome sedimentation and lower figures show that of ribo-T as described in Experimental procedures. Mg2+ concentration was 4 mM. B Complete splitting of ribosome by RRF is unnecessary for translational coupling; ribo-T functions in a similar manner to wild-type ribosomes. Incorporation of [14C]-Leucine coded for by CUG and CUA codons in frame with AUG was measured as in Fig 10B. The reaction mixture contained 7 mM Mg2+ where RRF requirement was more distinctly observed.
As shown in this figure, in the presence of RRF (open circles), the incorporation of labeled leucine (AUG frame) by ribo-T greatly increased compared with that in the absence of RRF (closed circles). This is similar to the observation in Fig 10B with wild-type ribosomes. This indicates that ribo-T was released from the mRNA at UAA of UAAUG by RRF and bound to AUG without complete splitting. We conclude that complete ribosome splitting is not an obligatory step during the ribosome recycling.
Discussion
All in vivo and in vitro observations are consistent with RRF-dependent release of ribosomes from mRNA during termination process—Prevention of unwanted protein synthesis by RRF
The role of RRF during the normal translation termination process in vivo has remained obscure despite extensive biochemical work on RRF. In this paper, we chose the translational coupling system without the SD sequence near the junction sequence as a model system to elucidate this problem. We conclude from these studies that the major function of RRF during the termination process is to release mRNA from the post termination complex (PoTC). We consider that the term “release” includes two stages, release to the immediate vicinity of the mRNA first, followed by the second stage where the ribosomes are far away from the mRNA. In the first stage, the released ribosomes are different from the free ribosomes in that they are close enough to the mRNA to rebind selectively to it without the SD sequence. In the translational coupling discussed in this paper, we deal mostly with the first stage of the release.
Here, we discuss the in vivo experiment (Fig 3 and Table 1) most directly indicating the release of ribosomes from mRNA by RRF. In the absence of tsRRF, at the non-permissive temperature (39°C), a much higher proportion of ribosomes from the u-ORF were engaged in downstream reading than those in the presence of RRF at the permissive temperature (28°C) (Table 1, All frames). Since ribosomes must stay on the mRNA for translation of downstream region, RRF-dependent release of ribosomes from mRNA of the PoTC would decrease the total number of ribosomes that read the downstream region. Saito et al., [48] reported that, upon RRF depletion by repressing its transcription, 70S ribosomes accumulated in 3’-UTR with ribosome profiling. However, when a reporter gene was connected downstream, they did not detect re-initiation of translation by these ribosomes. In contrast, we observed unscheduled translation as described in Fig 3 and the previous communication [16]. Although the reason of this discrepancy is not clear, it may be that the residual amount of RRF still remained after RRF depletion because protein synthesis remained as much as 40% [48]. It should be emphasized that, at the non-permissive temperature, over-all protein synthesis in LJ14 is severely reduced due to the lack of ribosome recycling for a new round of protein synthesis [49].
The in vitro experiments using wild-type RRF and the mRNA designed to study translational coupling (Fig 8) also suggested the ribosome releasing function of RRF. In this system, in the presence of RRF, ribosomes began to read from AUG of the junction sequence UAAUG. This means that the ribosome must be released from UAA. The released ribosomes must be rebound to mRNA to read the rest of the mRNA in frame with AUG (leucine incorporation, Fig 10A and 10B). In contrast, in the absence of RRF, the ribosome remained on UAA and translated downstream in frame with UAA (valine incorporation, Fig 11).
The release of ribosomes from PoTC results in the prevention of the synthesis of unwanted protein such as the peptide containing valine as shown in Fig 11. This is because this peptide is not scheduled to be synthesized and would have a lethal effect. Prevention of unscheduled translation is a consequence of the function of RRF; the release of mRNA from PoTC.
When ribosomes translating the u-ORF reach the junction sequence, the tRNA carrying the nascent u-ORF gene product is bound to the last codon of u-ORF at the P-site. The u-ORF peptide is then released by the release factor attracted by the termination codon at the A-site. The resulting ribosomal complex has the tRNA bound mostly to P/E site (some at the P/P site [24]). In Fig 13, we summarize RRF’s function in vivo during natural termination process represented by our model translational coupling system. In complex (a) of this figure, the A-site of PoTC is empty because the release factor which had hydrolyzed the ester bond between tRNA and the u-ORF protein was released with the help of RF3 [50,51]. This will give RRF the chance to bind at the A/P site [22,23], resulting in complex (b) formation. In the step from complex (b) to (c), tRNA is released from the P-site [24], the ribosome is released from the mRNA without splitting due to the action of RRF together with EF-G and GTP [1,41]. The released 70S ribosomes may or may not dissociate into 30S and 50S subunits by RRF and EF-G (or IF3 alone). At this point the recycling step is completed. The exact molecular mechanism of release of RRF and EF-G from PoTC is to be elucidated. In complex (c), because of the proximity of AUG to UAA, some of the released ribosomes which are in the immediate vicinity of mRNA are re-bound to AUG of UAAUG with formyl methionyl tRNA. Re-initiation of translation starts and the reporter lacZ gene is expressed. It should be noted that there are junction sequences with an SD sequence of the d-ORF [52]. However, such a system is not suitable for studying the behavior of ribosomes at the end of the natural ORF without an SD sequence around the termination codon.
Complex (a); Post Termination Complex (PoTC) at the junction sequence of translationally coupled ORFs. The junction sequence UAAUG is shown in bold. Complex (b); RRF is bound on the ribosome at A/P-site. Complex (c); Completed 70S initiation complex. Process from (b) to (c); The ribosome and deacylated tRNA are released from the mRNA by RRF and EF-G action. The ribosome may or may not split into subunits. Both the 70S ribosomes and the ribosome subunits that engaged in the reading of the u-ORF may participate in downstream reading. The released 70S ribosomes or the dissociated ribosome subunits bind to the adjacent AUG on the same mRNA with the help of fMet-tRNA.
Is the splitting of 70S ribosome essential for disassembly of PoTC?
Although our in vivo data presented in this paper do not directly exclude the possibility of 30S/mRNA complex as an intermediate of disassembly of PoTC, we avoid such complex. This is because none of the ribosomes from PoTC without SD near the termination triplet had 16S ribosomal RNA only. Additionally, none of the PoTC had visible poly-30S ribosomal subunits (Figs 5 and 9 of [24]) [28]. The 30S/mRNA complex have been observed only with those with nearby SD (for example [29]).
Furthermore, complete splitting of ribosomes is not necessary for translational coupling because ribosomes with tethered subunits [40] still function like wild-type ribosomes at the junction sequence. The observation in vitro (Fig 12B) showed that ribosome release from UAA of the junction sequence UAAUG and rebinding to AUG still occurred (resulting in leucine incorporation in frame with AUG) without complete separation of subunits. This is consistent with the notion that initiation with 70S ribosomes [53], rather than subunits, is commonly involved in prokaryotic protein synthesis [54]. Translational coupling, a common ribosomal activity [37], must also be occurring in this mutant [40]. These observations support scheme not involving 30S/mRNA intermediate. In conclusion, we presented data to emphasize that the major in vivo function of RRF in the normal termination process is to release ribosomes from the PoTC without splitting.
Effects of high culture temperature in the absence of RRF
In this paper, we used a tsRRF mutant to eliminate the function of RRF in vivo. With this mutant, high culture temperature and simultaneous loss of RRF created an unusual situation. Under these unique conditions, ribosomes at junction sequences exhibited unexpected behavior. The frameshift of ribosomes on mRNA to all frames (temperature-dependent frameshift) occurred (Figs 3 and 4). We should add that this frameshift is limited to the mRNA-bound ribosomes without peptidyl groups. Such ribosomal complexes are imaginable when RRF is inactivated because the substrate of RRF is the ribosomes with one tRNA without a peptidyl group mostly in the P/E site [24]. Because of the absence of the peptidyl group which stabilizes ribosomes on the mRNA, the ribosome would slide on the mRNA resulting in the thermal frameshift.
The high expression of d-ORF in Fig 6 panel (d) in the absence of RRF due to high culture temperature suggests that ribosome-bound mRNA can take on a secondary structure at the junction region. With short u-ORF, such secondary structures would be able to shorten the distance between SD and the termination codon of u-ORF. In the case of Fig 6 panel (d), by base-pairing between front and rear parts of the u-ORF, the SD sequence (AGGA) and the termination codon UAA would adjoin each other (hairpin structure 3 of Fig 14). This short distance between SD and UAA would stimulate u-ribosomes on UAA to bind to AUG of UAAUG resulting in high d-ORF translation. However, the idea of secondary structure formation is against the general belief that mRNA bound to the ribosome at the channel of the 30S subunit [55] cannot take on a secondary structure. Perhaps our assumption (the secondary structure formation of ribosome-bound mRNA) is only possible when the ribosome does not have a peptidyl group as in the case of PoTC.
Stem and loop structures were constructed according to Freier et al. [56]. The SD sequence (5’-AGGA-3’), initiation and termination codons are represented by capital letters. Termination codons of u-ORF are boxed for clarity. Bars between nucleotides represent Watson-Crick type base-pairing.
The influence of nearby SD on the role of RRF as a ribosome-releasing factor
During our studies, we noticed that the SD sequence of short u-ORF has a strong influence on the behavior of ribosomes in translational coupling. The SD of the short u-ORF attracted ribosomes which were released by RRF from the termination codon. This caused the ribosomes to bind back to the u-ORF initiation codon resulting in loss of d-ORF reading (for example, Fig 6 panels (b), (c) and (d)). Furthermore, nearby SD pulled even the mRNA-bound ribosomes that resulted from inactivation of temperature sensitive RRF (Fig 6 panel (b)). This is consistent with the report that SD has an inhibitory effect on movement of mRNA-bound translating ribosomes [57]. Several reports indicate that SD sequences facilitate frameshift at the region involving slippery sequences [58–60]. This is most likely due to the pulling force of SD sequences on mRNA-bound translating ribosomes.
We showed the lack of downstream reading when SD of u-ORF was near the junction sequence as discussed above. This indicates that the use of short mRNA with SD (quoted in the recent review [61]) should be avoided for studies of RRF action at the end of natural ORF. The only “natural” short mRNA (mini-gene) with SD we could find is mRNA encoding a dipeptide related to the lambda phage [62]. The expression of mini-genes encoding less than seven amino acids is toxic to growth of the cell due to accumulation of peptidyl-tRNA and depletion of the tRNA [63]. The toxicity increases as the length of mini-gene shortens [63]. Zavialov et al., [8] reported that the original interaction between SD and anti-SD remained intact in the PoTCs in vitro when the lengths of ORFs were shorter than 6 codons, making a small loop containing the SD sequence. Their results are consistent with our idea that SD pulls back ribosomes finishing translation of short u-ORF without d-ORF translation. These in vitro and in vivo studies predict that the use of short ORF would make it impossible to observe the release of ribosomes from the mRNA. In support of this finding, a cryo-electron microscopic study with short mRNA (4 codons ORF) containing SD showed that the mRNA is never released from the PoTC [29]. The in vitro studies with various short mRNAs suggest that the SD sequence of the short ORF pulls back ribosomes from the termination codon of the ORF toward the SD and binds them to the initiation codon of the ORF. Therefore, the behavior of the ribosome after finishing reading such a short ORF with SD may not necessarily be the same as that of ribosomes released from the termination codon of natural ORFs without nearby SD. Here, we developed a new in vitro translational coupling system with 027mRNA which was long enough to abolish the effect of SD on ribosomal behavior at the termination step. Using this system, we showed that RRF released ribosomes at the termination codon of the mRNA in vitro. The studies presented in this paper show the important function of RRF, release of ribosomes from mRNA, which could not be observed in the in vitro studies with short ORFs containing SD.
There are translational couplings with [36] and without SD of d-ORF [39,42]. Our results stated above are partially consistent with the recent publication by Saito et al., [48] which shows that loss of RRF does not affect translational coupling. This is because they used gene pairs having SD motifs (for example, AGGA) in the junction regions. We think that loss of RRF activity of releasing ribosomes from termination codon is compensated by pulling force of SD to ribosomes remaining at the termination codon. In the current study, we showed that, with the help of the SD of short u-ORF near the junction sequence, u-ribosomes could initiate translation of d-ORF even in the absence of RRF (Fig 7). On the contrary, in the absence of nearby SD, RRF is essential to initiate downstream translation from only the AUG of d-ORF (Fig 3) [39].
Materials and methods
Bacteria
Escherichia coli JM109 (recA1, endA1, gyrA96, thi, hsdR17, mcrA-, supE44, relA1, Δ(lac-proAB)/F’traD36, proAB+, lacIq, lacZΔM15) was obtained from Takara Bio Inc. and used as recipient of the plasmids for transformation. The E. coli strain LJ14/F’lacIq [39] is designated simply as LJ14 in this text.
Materials
Restriction endonucleases, DNA ligase, TaKaRa Ex Taq DNA polymerase and PrimeSTAR® Mutagenesis Basal Kit were purchased from Takara Bio Inc. The plasmids pUC18, pKK223-3 and pMC1871 DNAs were purchased from Pharmacia Biotech.
Plasmids
Plasmid pUC18 is a vector carrying the lac promoter and α-peptide sequence of the lacZ gene into which a 54 base pair multiple cloning site (MCS) polylinker sequence is inserted. Plasmid pKK223-3 is a vector carrying the tac promoter and a 33-base pair MCS polylinker sequence. Plasmid pMC1871 has the lacZ gene lacking its promoter, the ribosome-binding site, and the twenty-seven 5’-terminal nucleotides including the initiation codon AUG of the lacZ gene, which is represented as lacZ(Δ27) in this paper because of the lack of 27 nucleotides. The DNA sequences from the junction sequence through the reporter gene of the plasmids were confirmed using CEQ8000 DNA sequencer (Beckman Coulter, Inc.) or 3130 Genetic Analyzer (Applied Biosystems). Preparations of plasmids used in this study are described in Supplemental Information and their nucleotide sequences are shown in S1 Table.
Assay of β-galactosidase activity
E. coli LJ14 cells harboring the plasmid carrying the lacZ(Δ27) gene or the lacZ(Δ78) gene were grown at 28°C in LB broth (1.0% tryptone (Difco), 0.5% yeast extract, 1.0% sodium chloride) containing 50 μg/ml ampicillin to a cell density (OD660) of ~0.13. At that time, the culture was divided into two parts and 2 mM (final concentration) IPTG was added to each part to induce transcription of the lac gene. Then, one part was transferred and incubated at 39°C for an additional 2 hours. The other part was kept at 28°C. At the indicated times, an aliquot (100 μl) was taken and the β-galactosidase activity was measured according to Miller [64] except that an OD660 of cell density was used instead of OD600. Miller units were calculated using the conversion factor, OD660/OD600 = 0.57. The β-galactosidase activity was expressed as means of two or three experiments with ± SE shown by error bars. When the size of the bar was smaller than that of the circles the bar was not shown in the figure.
In vitro translation system using wild-type and tethered ribosomes
The reaction mixtures (210 μl) contained 4 or 7 mM magnesium acetate, 150 mM potassium chloride, 20 mM Tris-HCl pH 7.6, 300 μM spermidine, 15 mM putrescine, 1 mM dithiothreitol, 1 mM GTP, 2 mM ATP, 10 mM phosphoenolpyruvate (PEP), 0.07 mg/ml PEP Kinase, 5 μM EF-G, 1 μM of each initiation factor (IF1, IF2 and IF3), 10 μM EF-Tu, 5 μM EF-Ts, 140 U of aa-tRNA synthetase, 1 μM formyl methionine transferase, 850 μM 10-formyltetrahydrofolate, 2 μM RF1, 2 μM RF3, 20 μM of each amino acid required for the translation of 027mRNA [45], 10 μM tRNAs mixture, 0.5 μM ribosome, 2.5 μM 027mRNA and 5 μM RRF, when present. The reaction mixtures were incubated at 37°C and after 0, 5, 10, 15, 20 and 40 minutes of incubation, 30 μl of the reaction mixture was spotted on a 1 cm x 1 cm 3MM filter and dropped in 5% TCA and 0.25% sodium tungstate for at least 1 hour at room temperature. The TCA/sodium tungstate mixture was changed and boiled for 10 minutes, washed three times with the same mixture, one time with ethanol/ether (v/v) and one time with ether. The filters were dried and the radioactivity was measured with a scintillation counter. For measuring phenylalanine incorporation, unlabeled phenylalanine was replaced in the above reaction mixture with 0.2 μM [3H]-phenylalanine and 19.8 μM of unlabeled phenylalanine (final concentration was 20 μM). 20 pmols of phenylalanine gave 2,650 CPM. This value was used to calculate the pmol of phenylalanine incorporated. For measuring leucine incorporation, [14C]-leucine was added in the reaction mixture to a final concentration of 20 μM. Twenty pmols of leucine corresponded to 13,447 CPM. For measuring valine incorporation, [14C]-valine was added in the reaction mixture to a final concentration of 25 nM. Twenty pmols of valine corresponded to 15,600 CPM.
Sedimentation analysis
Wild-type ribosomes and ribo-T were each incubated at 30°C for two minutes with 5 μM EF-G and 5 μM IF3 in the presence or absence of 5 μM RRF. After incubation, the reaction mixture was placed on ice to stop the splitting reaction and loaded onto a 4.5 ml sucrose gradient (15–35%) and spun for 3 hours and 45 minutes at 40,000 RPM using a Beckman SW50.1 rotor.
Acknowledgments
The authors would like to thank Dr. Shura Mankin of UIC for sending us E. coli strain SQ171 containing the tethered ribosomes. We thank Dr. Gualerzi of University of Camelino, Italy for providing us 027DNA. Thanks also H. Chen of Thomas Jefferson University for his help in preparation of the manuscript. We thank Ms. Noriko Hakoshima of Teikyo University for helping to determine DNA sequences of plasmids used in this study.
References
- 1. Hirashima A, Kaji A. Purification and properties of ribosome-releasing factor. Biochemistry. 1972;11(22):4037–44. pmid:4563926.
- 2. Shimizu I, Kaji A. Identification of the Promoter Region of the Ribosome-Releasing Factor Cistron (frr). Journal of bacteriology. 1991;173(16):5181–7.
- 3. Janosi L, Hara H, Zhang S, Kaji A. Ribosome recycling by ribosome recycling factor (RRF)—An important but overlooked step of protein biosynthesis. Advances in biophysics. 1996;32:121–201. pmid:8781287.
- 4. Hirokawa G, Demeshkina N, Iwakura N, Kaji H, Kaji A. The ribosome-recycling step: consensus or controversy? Trends in biochemical sciences. 2006;31(3):143–9. pmid:16487710
- 5. Janosi L, Shimizu I, Kaji A. Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:4249–53. pmid:8183897
- 6. Ogawa K, Kaji A. Requirement for ribosome-releasing factor for the release of ribosomes at the termination codon. European journal of biochemistry / FEBS. 1975;58:411–9. pmid:1183444
- 7. Karimi R, Pavlov MY, Buckingham RH, Ehrenberg M. Novel roles for classical factors at the interface between translation termination and initiation. Molecular cell. 1999;3(5):601–9. Epub 1999/06/09. pmid:10360176.
- 8. Zavialov AV, Hauryliuk VV, Ehrenberg M. Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G. Molecular cell. 2005;18(6):675–86. Epub 2005/06/14. pmid:15949442.
- 9. Hirokawa G, Nijman RM, Raj VS, Kaji H, Igarashi K, Kaji A. The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits. Rna. 2005;11(8):1317–28. pmid:16043510
- 10. Peske F, Rodnina MV, Wintermeyer W. Sequence of steps in ribosome recycling as defined by kinetic analysis. Molecular cell. 2005;18(4):403–12. pmid:15893724.
- 11. Alhebshi A, Sideri TC, Holland SL, Avery SV. The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species. Molecular biology of the cell. 2012;23(18):3582–90. Epub 2012/08/03. pmid:22855532
- 12. Becker T, Franckenberg S, Wickles S, Shoemaker CJ, Anger AM, Armache JP, et al. Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature. 2012;482(7386):501–6. Epub 2012/02/24. pmid:22358840.
- 13. Nurenberg E, Tampe R. Tying up loose ends: ribosome recycling in eukaryotes and archaea. Trends in biochemical sciences. 2013;38(2):64–74. Epub 2012/12/26. pmid:23266104.
- 14. Young DJ, Guydosh NR, Zhang F, Hinnebusch AG, Green R. Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3’UTRs In Vivo. Cell. 2015;162(4):872–84. Epub 2015/08/16. pmid:26276635
- 15. Ryoji M, Berland R, Kaji A. Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor. Proceedings of the National Academy of Sciences of the United States of America. 1981;78(10):5973–7. pmid:7031641
- 16. Janosi L, Mottagui-Tabar S, Isaksson LA, Sekine Y, Ohtsubo E, Zhang S, et al. Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. EMBO J. 1998;17(4):1141–51. Epub 1998/03/28. pmid:9463391
- 17. Petrelli D, LaTeana A, Garofalo C, Spurio R, Pon CL, Gualerzi CO. Translation initiation factor IF3: two domains, five functions, one mechanism? EMBO J. 2001;20(16):4560–9. Epub 2001/08/14. pmid:11500382
- 18. Subramanian AR, Davis BD. Activity of initiation factor F3 in dissociating Escherichia coli ribosomes. Nature. 1970;228(5278):1273–5. Epub 1970/12/26. pmid:4922688.
- 19. Subramanian AR, Davis BD. Release of 70’S ribosomes from polysomes in Escherichia coli. J Mol Biol. 1973;74:45–56.
- 20. Vivanco-Dominguez S, Bueno-Martinez J, Leon-Avila G, Iwakura N, Kaji A, Kaji H, et al. Protein Synthesis Factors (RF1, RF2, RF3, RRF, and tmRNA) and Peptidyl-tRNA Hydrolase Rescue Stalled Ribosomes at Sense Codons. J Mol Biol. 2012;417(5):425–39. Epub 2012/02/14. pmid:22326347.
- 21. Selmer M, Al-Karadaghi S, Hirokawa G, Kaji A, Liljas A. Crystal Structure of Thermotoga maritima Ribosome Recycling Factor: a tRNA mimic. Science. 1999;286(5448):2349–52. pmid:10600747.
- 22. Lancaster L, Kiel MC, Kaji A, Noller HF. Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing. Cell. 2002;111(1):129–40. Epub 2002/10/10. pmid:12372306.
- 23. Agrawal RK, Sharma MR, Kiel MC, Hirokawa G, Booth TM, Spahn CMT, et al. Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(24):8900–5.
- 24. Iwakura N, Yokoyama T, Quaglia F, Mitsuoka K, Mio K, Shigematsu H, et al. Chemical and structural characterization of a model Post-Termination Complex (PoTC) for the ribosome recycling reaction: Evidence for the release of the mRNA by RRF and EF-G. PloS one. 2017;12(5):e0177972. pmid:28542628.
- 25. Pai RD, Zhang W, Schuwirth BS, Hirokawa G, Kaji H, Kaji A, et al. Structural insights into ribosome recycling factor interactions with the 70S ribosome. J Mol Biol. 2008;376(5):1334–47. pmid:18234219
- 26. Weixlbaumer A, Petry S, Dunham CM, Selmer M, Kelley AC, Ramakrishnan V. Crystal structure of the ribosome recycling factor bound to the ribosome. Nature structural & molecular biology. 2007;14(8):733–7. pmid:17660830.
- 27. Barat C, Datta PP, Raj VS, Sharma MR, Kaji H, Kaji A, et al. Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits. Molecular cell. 2007;27(2):250–61. pmid:17643374
- 28. Yokoyama T, Shaikh TR, Iwakura N, Kaji H, Kaji A, Agrawal RK. Structural insights into initial and intermediate steps of the ribosome-recycling process. EMBO J. 2012;31(7):1836–46. Epub 2012/03/06. pmid:22388519.
- 29. Fu Z, Kaledhonkar S, Borg A, Sun M, Chen B, Grassucci RA, et al. Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Structure. 2016;24(12):2092–101. pmid:27818103
- 30. Hirashima A, Kaji A. Role of elongation factor G and a protein factor on the release of ribosomes from messenger ribonucleic acid. The Journal of biological chemistry. 1973;248(21):7580–7. Epub 1973/11/10. pmid:4583357.
- 31. Savelsbergh A, Rodnina MV, Wintermeyer W. Distinct functions of elongation factor G in ribosome recycling and translocation. Rna. 2009;15(5):772–80. pmid:19324963
- 32. Borg A, Pavlov M, Ehrenberg M. Complete kinetic mechanism for recycling of the bacterial ribosome. Rna. 2016;22(1):10–21. pmid:26527791.
- 33. Chen Y, Kaji A, Kaji H, Cooperman BS. The kinetic mechanism of bacterial ribosome recycling. Nucleic acids research. 2017;45(17):10168–77. pmid:28973468.
- 34. Prabhakar A, Capece MC, Petrov A, Choi J, Puglisi JD. Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling. Cell reports. 2017;20(1):161–72. Epub 2017/07/07. pmid:28683310
- 35. Zhou D, Tanzawa T, Lin J, Gagnon MG. Structural basis for ribosome recycling by RRF and tRNA. Nature structural & molecular biology. 2020;27(1):25–32. Epub 2019/12/25. pmid:31873307.
- 36. Platt T, Yanofsky C. An intercistronic region and ribosome-binding site in bacterial messenger RNA. Proceedings of the National Academy of Sciences of the United States of America. 1975;72(6):2399–403. pmid:1094468
- 37. Salgado H, Moreno-Hagelsieb G, Smith TF, Collado-Vides J. Operons in Escherichia coli: genomic analyses and predictions. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(12):6652–7. pmid:10823905.
- 38. Oppenheim DS, Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980;95(4):785–95. Epub 1980/08/01. pmid:6162715
- 39. Inokuchi Y, Hirashima A, Sekine Y, Janosi L, Kaji A. Role of ribosome recycling factor (RRF) in translational coupling. EMBO J. 2000;19(14):3788–98. pmid:10899132
- 40. Orelle C, Carlson ED, Szal T, Florin T, Jewett MC, Mankin AS. Protein synthesis by ribosomes with tethered subunits. Nature. 2015;524(7563):119–24. pmid:26222032.
- 41. Hirashima A, Kaji A. Factor-dependent release of ribosomes from messenger RNA. Requirement for two heat-stable factors. J Mol Biol. 1972;65(1):43–58. pmid:4553259.
- 42. Ivey-Hoyle M, Steege DA. Translation of phage-f1 gene-VII occurs from an inherently defective initiation site made functional by coupling. J Mol Biol. 1989;208:233–44. pmid:2788746
- 43. Inokuchi Y, Takahashi R, Hirose T, Inayama S, Jacobson AB, Hirashima A. The Complete Nucleotide Sequence of the Group II RNA Coliphage GA. J Biochem. 1986;99:1169–80. pmid:3711059
- 44. Dunkle JA, Wang L, Feldman MB, Pulk A, Chen VB, Kapral GJ, et al. Structures of the bacterial ribosome in classical and hybrid states of tRNA binding. Science. 2011;332(6032):981–4. Epub 2011/05/21. 332/6032/981 pmid:21596992.
- 45. Brandi L, Fabbretti A, Milon P, Carotti M, Pon CL, Gualerzi CO. Methods for identifying compounds that specifically target translation. Methods in enzymology. 2007;431:229–67. pmid:17923238.
- 46. Shimizu Y, Kanamori T, Ueda T. Protein synthesis by pure translation systems. Methods. 2005;36(3):299–304. pmid:16076456.
- 47. Petrov AS, Bernier C, Hsiao C, Okafor CD, Tannenbaum ED, Stern J, et al. RNA-Magnesium-Protein Interactions in Large Ribosomal Subunit. The journal of physical chemistry B. 2012;116(28): 8113–20. Epub 2012/06/21. pmid:22712611.
- 48. Saito K, Green R, Buskirk AR. Ribosome recycling is not critical for translational coupling in Escherichia coli. eLife. 2020;9. Epub 2020/09/24. pmid:32965213
- 49. Hirokawa G, Inokuchi H, Kaji H, Igarashi K, Kaji A. In vivo effect of inactivation of ribosome recycling factor—Fate of ribosomes after unscheduled translation downstream of open reading frame. Molecular microbiology. 2004;54(4):1011–21.
- 50. Pavlov MY, Freistroffer DV, Heurgué-Hamard V, Buckingham RH, Ehrenberg M. Release factor RF3 abolishes competition between release factor RF1 and ribosome recycling factor (RRF) for a ribosome binding site. J Mol Biol. 1997;273(2):389–401. pmid:9344747.
- 51. Grentzmann G, Kelly PJ, Laalami S, Shuda M, Firpo MA, Cenatiempo Y, et al. Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex. Rna. 1998;4(8):973–83. pmid:9701288
- 52. Yanofsky C, Platt T, Crawford IP, Nichols BP, Christie GE, Horowitz H, et al. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic acids research. 1981;9(24):6647–68. Epub 1981/12/21. pmid:7038627
- 53. Moll I, Hirokawa G, Kiel MC, Kaji A, Blasi U. Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs. Nucleic acids research. 2004;32(11):3354–63. Epub June 23, 2004. pmid:15215335.
- 54. Yamamoto H, Wittek D, Gupta R, Qin B, Ueda T, Krause R, et al. 70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(9):E1180–9. pmid:26888283
- 55. Yusupova GZ, Yusupov MM, Cate JH, Noller HF. The path of messenger RNA through the ribosome. Cell. 2001;106(2):233–41. pmid:11511350
- 56. Freier SM, Kierzek R, Jaeger JA, Sugimoto N, Caruthers MH, Neilson T, et al. Improved free-energy parameters for predictions of RNA duplex stability. Proceedings of the National Academy of Sciences of the United States of America. 1986;83(24):9373–7. Epub 1986/12/01. pmid:2432595
- 57. Li GW, Oh E, Weissman JS. The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature. 2012;484(7395):538–41. Epub 2012/03/30. pmid:22456704
- 58. Bock LV, Caliskan N, Korniy N, Peske F, Rodnina MV, Grubmuller H. Thermodynamic control of -1 programmed ribosomal frameshifting. Nature communications. 2019;10(1):4598. Epub 2019/10/12. pmid:31601802
- 59. Yan S, Wen JD, Bustamante C, Tinoco I Jr., Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways. Cell. 2015;160(5):870–81. pmid:25703095
- 60. Larsen B, Wills NM, Gesteland RF, Atkins JF. rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift. Journal of bacteriology. 1994;176:6842–51. pmid:7961443
- 61. Rodnina MV. Translation in Prokaryotes. Cold Spring Harbor perspectives in biology. 2018;10(9). Epub 2018/04/18. pmid:29661790
- 62. Hernandez-Sanchez J, Valadez JG, Herrera JV, Ontiveros C, Guarneros G. lambda bar minigene-mediated inhibition of protein synthesis involves accumulation of peptidyl-tRNA and starvation for tRNA. EMBO J. 1998;17(13):3758–65. pmid:9649445
- 63. Heurgue-Hamard V, Dincbas V, Buckingham RH, Ehrenberg M. Origins of of minigene-dependent growth inhibition in bacterial cells. Embo J. 2000;19(11):2701–9. pmid:10835367.
- 64.
Miller JH. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escheichia coli and Related Bacteria. Illustrated ed. Miller JH, editor. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1992. 456 p.