Figure 1.
Secondary structure of a trans-splicing group I intron ribozyme.
The ribozyme (black) is base-paired to the target site of the mutated CAT mRNA (red). The 5′-splice site and 3′-splice site are indicated with arrowheads. During the trans-splicing reaction (arrows from left to right) the 3′-exon of the ribozyme (blue) replaces the 3′-portion of the mutated CAT mRNA, leading to the repaired CAT mRNA (red/blue). The positions of the P8 and P6b stem-loops in the ribozyme are indicated, together with the four positions 236, 238, 239, and 241.
Figure 2.
In vivo evolution of trans-splicing ribozymes.
A, Library plasmid used in the evolution, expressing the mutated CAT mRNA (CATMUT, red) under the control of a constitutive promoter (filled triangle), with a frameshift mutation (x). The ribozyme (black) is expressed from an inducible promoter (open triangle) and is linked to its 3′-exon (blue). B, Flow-chart of events in a single evolution cycle. C, Succession of events during the 21 cycles of evolution. Evolutionary cycles with mutagenic PCR are labeled with triangles, cycles with recombination are labeled with filled squares, and enrichment cycles are labeled with empty circles. Empty, grey, and black triangles correspond to a low, medium, and high mutagenesis rate, respectively. D, Increase in the number of mutations over the course of the evolution. The average number of mutations per ribozyme sequence is given. Error bars denote the standard error of the mean (n = 10). Symbols are as in (C). E, Increase in the activity of the pool during the evolution. The percentage of viable cells growing on plates with 20 µg/mL chloramphenicol is given. Symbols are the same as in (C).
Figure 3.
Enrichment of specific mutations during the evolution.
A, The number of mutations detected over all 21 cycles of evolution is plotted for each of the 414 nucleotide positions in the ribozyme. The graph represents a total of 2438 mutations. The structural elements corresponding to the nucleotide positions according to [51] are given at the top. The short helices P3, P7, and P9.0 are not labeled due to space constraints. The position of the P6b loop is indicated by an empty triangle. B, The frequency of mutations that appeared at least 15 times in total is shown over the cycles of evolution. The nucleotide position of these mutations is given in the left-most column. Ten sequences were obtained for each cycle, with blue denoting at least 2 mutations and red denoting at least four mutations detected for that cycle and nucleotide position. For cycles 3–8, where the evolution was split into two branches, the color-coding is averaged over both branches. C, The sequencing chromatogram of the evolved plasmid library after cycle 21 shows the enrichment of mutations 238, 239, and 240 in the ribozyme population (top), compared to the parent sequence (bottom). Note that at position 236, “C” (blue) represents only a small proportion of the evolved pool.
Figure 4.
Increased resistance to chloramphenicol in cells expressing the most efficient ribozyme isolated from the evolution.
The doubling time of E.coli cells in medium containing chloramphenicol is plotted as function of chloramphenicol concentration. The evolved ribozyme (triangles) facilitated shorter doubling times than the parent ribozyme (circles). Note that the horizontal scale is broken to denote the doubling time of both constructs in the absence of chloramphenicol. Error bars denote standard deviations from the geometric means of at least three independent experiments. If error bars are not visible they are smaller than the symbols.
Figure 5.
Identification of ribozyme mutations that increase chloramphenicol resistance.
A, Growth rates of E.coli cells containing different ribozyme variants, in medium containing 20 µg/mL chloramphenicol. The parent ribozyme (0 Mut), the most efficient, evolved ribozyme (12 Mut; black), and ribozyme M4 (4 Mut; green) are labeled with the number of mutations distant from the parent ribozyme. The label P8 (orange column) describes the ribozyme where the four mutations were transplanted from the P6b loop to the P8 loop of the parent ribozyme. All other variants (grey columns) are based on the 12-mutation ribozyme and show the effect of individual, labeled reversion mutations to the parent sequence. The absence of a column means that the doubling time was larger than 100 minutes in all experiments. Error bars are standard deviations from at least three experiments. B, Secondary structure of the ribozyme with the positions of the 12 mutations in (A) indicated by gray circles. The four beneficial mutations in the P6b stem-loop are shown in green.
Figure 6.
Identification of an E.coli protein that specifically interacts with the evolved P6b stem-loop.
A, Secondary structure of two 5′-biotinylated RNA hairpins that were used in the pull-down experiments. The four evolved, beneficial mutations of M4 are labeled with arrowheads. B, Silverstained SDS-polyacrylamide gel with proteins that were pulled down by the biotinylated hairpins. The size of marker proteins in kDa is indicated on the left. The area of the excised gel fragments at ∼50 kDa is indicated by black corners. C, Proteins that were identified by mass spectrometry from the excised gel fragments. Given is the number of peptides that were identified for the respective proteins with at least 95% confidence. The three columns for each experiment (parent, M4, beads) show the results from experiments with three biological replicates. Results for the transcription termination factor Rho are shown in bold.
Figure 7.
Effects of the M4 mutations on ribozyme function in vitro and in vivo.
The values on top of the graphs show the -fold increase of activity from parent ribozyme (white columns) to the M4 ribozyme (gray columns). A, Trans-splicing efficiency in vitro. The graph shows the percent of CATMUT RNA repaired within 3 hours of incubation. B, Trans-splicing efficiency in cells. Shown is the number of molecules of CATMUT RNA, ribozyme (Rz), and CATREP RNA per E.coli cell, as calculated from RT-qPCR experiments with total RNA. C, Enzymatic activity of CAT enzyme in E.coli cell lysates. Error bars are standard deviations from three biological replicates. D, Abundance of RNAs on ribosomes from E.coli strains expressing the parent ribozyme (white columns) or the ribozyme with four mutations M4 (grey columns). Shown is the percentage of RNAs detected in all fractions of the sucrose gradient, that were found in the polysome fractions. The -fold values above the graph show the increased loading of the respective RNA on polysomes for the M4 ribozyme versus the parent ribozyme. Error bars show standard deviations from three biological replicates.
Figure 8.
Frequency of oligo(C) sequences in the genome of E.coli.
The graph shows the relative abundance of homopolymers in the genome of E.coli, as a function of the length of the homopolymer sequence. Note that the values for oligo(C) (filled, black squares) and oligo(G) (filled, black circles) show the frequency of oligo(C) on the top and bottom strand of the genome, respectively. The values for oligo(A) and oligo(T) (filled triangles) are so similar that they cannot be distinguished in this graph. As comparison, the average of all triplet codons (white circle with standard deviation as error bars) and the frequency of the stop codon TAG and its reverse complement CTA (circles with grey filling) are shown. The genome used for this analysis was from E.coli K12 sub-strain MG1655 (gi|49175990).