Fig 1.
A. Linear genome organization of phage Mu. The middle gene product C (shown in Green and represented as dimer) activates transcription from the late gene promoters lys, I, P and mom. B. Mu late promoter sequences. Late promoter sequences were aligned with respect to their transcription start sites as determined by S1 nuclease mapping (Bolker, et al., 1989, Margolin, et al., 1989), and similarities in -10 and -35 regions. Heavy black line indicates the C-foot print in lys and mom promoters. A 10bp match between lys and I promoters and the 6T stretch common between lys and mom promoters in the spacer region are underlined in green and magenta respectively. Sequences of non-consensus -10 (boxed) and -35 elements (underlined in blue) are depicted.
Fig 2.
C is essential for transactivation from late promoters.
In vitro transcription assays carried out on lys,I,P (lanes 1–6), and at Pmom (lanes 9–10) promoters in the absence and presence of transactivator C. At mutant mom promoter—Ptin7, RNAP recruitment is C independent. The basal transcription in the absence of C is enhanced in its presence due to second step activation described in the text (lanes 7–8).
Fig 3.
C protein is not essential for recruitment of RNAP at lys, I and P promoters.
5′ P32 labeled lys, I, P, tin7 promoter constructs were incubated with increasing concentration of RNAP either in the absence or presence of C protein on ice for 10 min and samples were analyzed on 3.5% native PAGE at 4°C. The amount of free DNA [D] and RNAP-bound promoter DNA [DP] were quantified to determine the KB of RNAP binding. DP:D values were plotted as a function of RNAP concentration. A-D represent- the promoter binding affinity of RNAP in the absence and presence of C protein at lys, I, P and tin7 promoters respectively. RNAP binds these promoters irrespective of the presence of C protein. The results are representative of three independent experiments.
Fig 4.
C enhances the isomerization of closed complex to promoter open complex.
Open complex formation assays were carried out on 5′ end labeled promoter constructs as described in Experimental procedures. C was added to the reaction wherever indicated. RNAP-promoter open complexes [RPO] were resolved on 3.5% native page. A-C indicate the requirement of C protein for effective open complex formation at lys,I,P promoters respectively. D. RPo formation on tin7 promoter is C- independent. The open complexes were quantified using Multi gauge software. The complex formed at each promoter with RNAP in presence of C was taken as 100% and values were normalized accordingly. The results are an average of three independent experiments.
Fig 5.
Transactivator C facilitates productive transcription at lys, I, P promoters.
Abortive transcription profile of lys, I, P promoters. Low amounts of abortive transcripts are seen at lys, I, P in the absence of C protein but no productive transcription is observed. In the presence of C, though the abortive transcription is seen, RNAP enters into productive elongation phase and transcripts are synthesized.
Fig 6.
G524DrpoCRNAP exhibits defective transcription from other late promoters similar to that observed at Pmom.
In vitro transcription assays were carried out on phage Mu late promoter constructs (lys,I,P, mom and mutant mom promoter-tin7) and E. coli 70 promoter T7A1. A) Samples were analyzed on 8% denaturing PAGE to assess the productive transcripts and quantified using Muti gauge software. Transcription at each individual promoter with WT RNAP was taken as 100% and the values were normalized. B) Bar diagram depicts quantitative representation of transcription efficiency. G524D RNAP showed reduced productive transcription at all C- dependent promoters compared to WT RNAP but was competent at T7A1 promoter. The results are an average of three independent experiments.
Fig 7.
Differential activation by C at late promoters.
Cartoonic representation of the mechanism of C mediated transactivation at late promoters. C activates transcription by a multi-step mechanism at Pmom. C acts initially to recruit RNAP to the mom promoter and then facilitates promoter escape. At promoters lys, I and P- C acts at a single step and facilitates open complex formation which proceeds into productive elongation mode. In the absence of activator C, the open complex formed remains transcriptionally inactive. RNAP holoenzyme—magenta, C dimer-green oval, out of phase promoter elements at Pmom is represented on DNA as blue and red rectangles respectively. CBS (C-binding site) over lapping -35 element is shown as green rectangle. CBS on Plys,PI,PP promoters is depicted as red rectangle. RNAP open complex at lys, I, P promoters is shown as open circle.
Table 1.
Oligonucleotides used in the study.