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Ribosome Collisions Alter Frameshifting at Translational Reprogramming Motifs in Bacterial Mrnas

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Ribosome Collisions Alter Frameshifting at Translational Reprogramming Motifs in Bacterial Mrnas Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs Angela M. Smitha, Michael S. Costelloa, Andrew H. Kettringa, Robert J. Wingoa, and Sean D. Moorea,1 aBurnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816 Edited by Dieter Söll, Yale University, New Haven, CT, and approved September 12, 2019 (received for review June 20, 2019) Translational frameshifting involves the repositioning of ribosomes P- and A-sites, but the molecules tilt such that the P-site tRNA’s on their messages into decoding frames that differ from those dic- acceptor end enters the exit site (E-site) and the A-site tRNA’s tated during initiation. Some messenger RNAs (mRNAs) contain mo- acceptor end enters the P-site. This rearrangement is accompa- tifs that promote deliberate frameshifting to regulate production of nied by a movement of the uL1 stalk to partially close the tRNA the encoded proteins. The mechanisms of frameshifting have been E-site (7). At this stage, the ribosome binds to elongation factor investigated in many systems, and the resulting models generally G (EF-G), which couples the energy of guanosine triphosphate involve single ribosomes responding to stimulator sequences in their (GTP) hydrolysis to promote a transient rotation of the small sub- engaged mRNAs. We discovered that the abundance of ribosomes unit and to drive a 3-nucleotide ribosome translocation event. on messages containing the IS3, dnaX,andprfB frameshift motifs After translocation, the tRNA that was originally in the P-site significantly influences the levels of frameshifting. We show that temporarily resides in the E-site, the peptidyl-tRNA is fully po- this phenomenon results from ribosome collisions that occur during sitioned in the P-site, and the ribosome returns to a relaxed, translational stalling, which can alter frameshifting in both the stalled nonrotated state awaiting a new aa-tRNA match in the A-site (3). and trailing ribosomes. Bacteria missing ribosomal protein bL9 are If there is a weak interaction between the tRNAs and the known to exhibit a reduction in reading frame maintenance and to mRNA during translocation, a translational frameshift can occur if have a strong dependence on elongation factor P (EFP). We discov- there is an alternative base pairing option in the vicinity (1). ered that ribosomes lacking bL9 become compacted closer together Likewise, if there is a delay in decoding or if there is mechanical during collisions and that the E-sites of the stalled ribosomes appear stress on the ribosome from a nearby mRNA secondary structure, MICROBIOLOGY to become blocked, which suggests subsequent transpeptidation in ribosomes can frameshift or hop over an mRNA segment (1). transiently stalled ribosomes may become compromised in the absence Aside from A- and P-site interactions, a cognate deacylated tRNA of bL9. In addition, we determined that bL9 can suppress frameshifting in the E-site can reduce frameshifting while it remains base-paired of its host ribosome, likely by regulating E-site dynamics. These to the mRNA (8–12). In bacteria, allostery between the E-sites and findings provide mechanistic insight into the behavior of colliding A-sites has been observed during early translation cycles, in that E- ribosomes during translation and suggest naturally occurring frameshift site tRNAs are retained until A-site tRNAs are delivered (12). elements may be regulated by the abundance of ribosomes relative This synchrony is reduced once the nascent peptide reaches to an mRNA pool. ∼4 amino acids long, and bacterial ribosomes switch to discarding E-site tRNAs irrespective of A-site occupancy (12, 13). In contrast ribosome | translation | frameshift | bL9 | dnaX to earlier models, it is now believed there is no connection between E-site occupancy and the fidelity of A-site tRNA selection (14). aturally occurring translational frameshift motifs generally However, recent evidence suggests that an incoming mRNA Ninclude a “slippery” messenger RNA (mRNA) sequence that contains an out-of-frame alternate transfer RNA (tRNA)−mRNA Significance pairing option and adjacent stimulatory elements that interact with the ribosome to promote transient stalling or unseating (1). Although these features are clearly validated experimentally, much Ribosomes move along mRNAs in 3-nucleotide steps as they in- of the translation fidelity literature focuses on the behavior of terpret codons that specify which amino acid is required at each ribosomes in isolation. Here, we show that ribosome collisions position in the protein. There are multiple examples of genes with induced by translational stalling should also be considered as part DNA sequences that do not match the produced proteins because of these frameshifting mechanisms and that ribosome collisions ribosomes move to a new reading frame in the message before and overcompaction of polysomes may interfere with ribosome finishing translation (so-called frameshifting). This report shows function. that, when ribosomes stall at mRNA regions prone to cause The translation of codons within mRNA open reading frames frameshifting events, trailing ribosomes that collide with them (ORFs) is now understood in substantial detail (reviewed in refs. 2 can significantly change the outcome and potentially regulate and 3). Upstream of many bacterial ORFs, a short Shine−Dalgarno protein production. This work highlights the principle that biological macromolecules do not function in isolation, and it provides an (SD) sequence is present that is complementary to a portion of the example of how physical interactions between neighboring com- small ribosomal subunit RNA (4). During translation initiation, plexes can be used to augment their performance. an interaction between these RNA segments helps to position the start codon in the peptidyl site (P-site) of the assembling ribo- Author contributions: A.M.S. and S.D.M. designed research; A.M.S., M.S.C., A.H.K., and some, and the strength of complementarity can substantially alter R.J.W. performed research; A.H.K. contributed new reagents/analytic tools; A.M.S., M.S.C., the rate of translation initiation (5, 6). During translation elongation, A.H.K., and S.D.M. analyzed data; and A.M.S. and S.D.M. wrote the paper. codons in the adjacent A-site are evaluated for complementarity The authors declare no competing interest. to the anticodon stems of aminoacylated tRNAs (aa-tRNA). When This article is a PNAS Direct Submission. a match is found, the ribosome permits a chemical reaction be- This open access article is distributed under Creative Commons Attribution-NonCommercial- tween the amino acid on the A-site tRNA and the acyl bond that NoDerivatives License 4.0 (CC BY-NC-ND). connects the nascent peptide to the P-site tRNA, thereby transferring 1To whom correspondence may be addressed. Email: [email protected]. the protein chain to the A-site tRNA. This reaction causes the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2 tRNAs to shift their orientations into the P/E A/P hybrid state, 1073/pnas.1910613116/-/DCSupplemental. wherein the anticodon regions of the tRNAs remain in the www.pnas.org/cgi/doi/10.1073/pnas.1910613116 PNAS Latest Articles | 1of11 Downloaded by guest on September 28, 2021 secondary structure can alter the small subunit conformation position of clamp loaders) and transposition (by altering the pro- and tRNA ejection rate from the E-site (15, 16). duction of transposases). Most naturally occurring frameshift motifs contain adjacent stimulator elements that encourage ribosomes to relocate. For Results example, the IS3 insertion element family contains a slippery sequence A High Ribosome Load Reduces Frameshifting at the IS3 Pseudoknot. and a downstream mRNA pseudoknot to induce −1 frameshifting We evaluated the influence of ribosome abundance on −1frame- during production of their transposases (17–19). The dnaX shifting using a well-characterized motif derived from insertion gene in many bacteria contains a −1 frameshift element that is element IS3 that contains a slippery tetrad sequence (A_AAG) stimulated by both an upstream SD-like sequence and a down- upstream of a pseudoknot stimulator (Fig. 1A). A prior study stream mRNA stem loop to control the ratio of Tau and Gamma reported that this motif induced substantial −1 frameshifting, proteins in the clamp loader complex of the DNA replisome (20– which was dependent on both the sequence of the slippery patch 22). In another example, the frameshift element in the prfB gene and the presence of the pseudoknot (19). We placed this motif in uses an upstream SD-like stimulator and transient stalling at a reporter plasmids that encoded a fluorescent protein (mOrange2) UGA stop codon to enhance +1 frameshifting (23–25). In each followed by the IS3 stimulator; upon −1 frameshifting, a larger example, ribosomes lose their grip on the mRNA and become fusion protein was generated that contained an appended FLAG repositioned in an alternate reading frame prior to resuming epitope (Fig. 1A). Thus, the ratio of the 2 protein forms served as a translation. readout of −1 frameshifting. These reporters also encoded ClpXP During a study of an extreme ribosome hopping event that oc- protease degrons in the −1and+1 frames preceding the test re- ’ curs during translation of bacteriophage T4 s gene 60 (26), it was gion, so that any frameshifted products unrelated to the test region ’ discovered that protein bL9 of the bacterial ribosome slarge would not accumulate (45, 46). Control reporters included a ver- subunit plays a role in maintaining translation fidelity (27). Since sion that lacked the slippery sequence and a version encoding the that discovery, several studies revealed that a lack of bL9 causes full-length fusion protein in frame. increased frameshifting, misincorporation, and ribosome hopping Cultures containing these plasmids were grown to early expo- in a number of scenarios (1, 28, 29).
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