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      Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs

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          Significance

          Ribosomes move along mRNAs in 3-nucleotide steps as they interpret codons that specify which amino acid is required at each position in the protein. There are multiple examples of genes with DNA sequences that do not match the produced proteins because ribosomes move to a new reading frame in the message before finishing translation (so-called frameshifting). This report shows that, when ribosomes stall at mRNA regions prone to cause frameshifting events, trailing ribosomes that collide with them can significantly change the outcome and potentially regulate protein production. This work highlights the principle that biological macromolecules do not function in isolation, and it provides an example of how physical interactions between neighboring complexes can be used to augment their performance.

          Abstract

          Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

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          Most cited references59

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          Structures of the bacterial ribosome at 3.5 A resolution.

          We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.
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            Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates.

            We have used two-dimensional polyacrylamide gel electrophoresis to fractionate tRNAs from Escherichia coli. A sufficiently high degree of resolution was obtained for 44 out of 46 tRNA species in E. coli to be resolved into individual electrophoretic components. These isolated components were identified by hybridization to tRNA-specific oligonucleotide probes. Systematic measurements of the abundance of each individual tRNA isoacceptor in E. coli, grown at rates varying from 0.4 to 2.5 doublings per hour, were made with the aid of this electrophoretic protocol. We find that there is a biased distribution of the tRNA abundance at all growth rates, and that this can be roughly correlated with the values of codon frequencies in the mRNA pools calculated for bacteria growing at different rates. The tRNA species cognate to abundant codons increase in concentration as the growth rate increases but not as dramatically as might be anticipated. The levels of most of the tRNA isoacceptors cognate to less abundant codons remain unchanged with increasing growth rates. The result of these changes in tRNA abundance is that the relative increase in the amounts of major tRNA species in the bacteria growing at the fastest growth rates is more modest than previous estimates from this laboratory suggested. Furthermore, a systematic error in previous estimates of ribosomal RNA content of the bacteria has been detected. This will account for the quantitative discrepancies between the previous and the present data for tRNA abundance.
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              EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.

              Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                22 October 2019
                7 October 2019
                7 October 2019
                : 116
                : 43
                : 21769-21779
                Affiliations
                [1] aBurnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, FL 32816
                Author notes
                1To whom correspondence may be addressed. Email: Sean.Moore@ 123456ucf.edu .

                Edited by Dieter Söll, Yale University, New Haven, CT, and approved September 12, 2019 (received for review June 20, 2019)

                Author contributions: A.M.S. and S.D.M. designed research; A.M.S., M.S.C., A.H.K., and R.J.W. performed research; A.H.K. contributed new reagents/analytic tools; A.M.S., M.S.C., A.H.K., and S.D.M. analyzed data; and A.M.S. and S.D.M. wrote the paper.

                Author information
                http://orcid.org/0000-0003-1606-7771
                Article
                201910613
                10.1073/pnas.1910613116
                6815119
                31591196
                8a21ee78-b753-4999-aa4a-b0284f2a5d6f
                Copyright © 2019 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 11
                Funding
                Funded by: HHS | NIH | National Institute of General Medical Sciences (NIGMS) 100000057
                Award ID: 1R01GM118896
                Award Recipient : Angela M Smith Award Recipient : Michael S. Costello Award Recipient : Andrew H. Kettring Award Recipient : Robert J Wingo Award Recipient : Sean D Moore
                Categories
                PNAS Plus
                Biological Sciences
                Microbiology
                PNAS Plus

                ribosome,translation,frameshift,bl9,dnax
                ribosome, translation, frameshift, bl9, dnax

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