Systematic analysis of nonprogrammed frameshift suppression in E. coli via translational tiling proteomics.

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.

Rhodobacter sphaeroides CarD Negatively Regulates Its Own Promoter.

Bioinformatic analysis showed previously that a majority of promoters in the photoheterotrophic alphaproteobacterium Rhodobacter sphaeroides lack the thymine at the last position of the -10 element (-7T), a base that is very highly conserved in promoters in bacteria other than alphaproteobacteria. The absence of -7T was correlated with low promoter activity using purified R. sphaeroides RNA polymerase (RNAP), but the transcription factor CarD compensated by activating almost all promoters lacking -7T tested in vitro, including rRNA promoters. Here, we show that a previously uncharacterized R. sphaeroides promoter, the promoter for carD itself, has high basal activity relative to other tested R. sphaeroides promoters despite lacking -7T, and its activity is inhibited rather than activated by CarD. This high basal activity is dependent on a consensus-extended -10 element (TGn) and specific features in the spacer immediately upstream of the extended -10 element. CarD negatively autoregulates its own promoter by producing abortive transcripts, limiting promoter escape, and reducing full-length mRNA synthesis. This mechanism of negative regulation differs from that employed by classical repressors, in which the transcription factor competes with RNA polymerase for binding to the promoter, and with the mechanism of negative regulation used by transcription factors like DksA/ppGpp and TraR that allosterically inhibit the rate of open complex formation. IMPORTANCE R. sphaeroides CarD activates many promoters by binding directly to RNAP and DNA just upstream of the -10 element. In contrast, we show here that CarD inhibits its own promoter using the same interactions with RNAP and DNA used for activation. Inhibition results from increasing abortive transcript formation, thereby decreasing promoter escape and full-length RNA synthesis. We propose that the combined interactions of RNAP with CarD, with the extended -10 element and with features in the adjacent -10/-35 spacer DNA, stabilize the promoter complex, reducing promoter clearance. These findings support previous predictions that the effects of CarD on transcription can be either positive or negative, depending on the kinetic properties of the specific promoter.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation.

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides rRNA promoters was unexpectedly weak, correlating with absence of -7T, the very highly conserved thymine found at the last position in -10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position -7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that R. sphaeroides RNAP can utilize -7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in ß- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked -7T, whereas it had no effect on three of the four native promoters that contained -7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained -7T, whereas 90 to 99% of promoters from non-α-proteobacteria contained -7T. Thus, promoters lacking -7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.