Evidence that the bypassing ribosome travels through the coding gap

Zwieb

2005

RNA

In bacteria, translation of mRNAs lacking stop codons produces truncated polypeptides and traps ribosomes in unproductive complexes. Potentially harmful truncated proteins are tagged with short peptides encoded by the mRNA-like domain of tmRNA and targeted for digestion by housekeeping proteases. We show that altered Escherichia coli transfer-messenger RNAs (tmRNAs) produce in vivo fusion proteins with peptide tags that extend far beyond the conventional termination signal of the wild-type tmRNA. Regions of tmRNA capable of serving as templates for protein synthesis include helix 5, as well as pseudoknots 2, 3, and 4. The removal of all six in-frame stop codons negatively affects tmRNA processing, thereby preventing translation of the 3 portion of the tRNA-like domain. These findings provide evidence that trans-translation can be accompanied by the unfolding of significant portions of the tmRNA molecule. Many of these conformational changes are likely to be required during transtranslation to maintain the ribosomal subunits in close proximity to the tmRNA for monitoring its transit.

“…2E). Our proposal is reminiscent of the recent demonstration that the 50-nt bypassed region of the T4 gene 60 mRNA is also unfolded (Gallant et al 2003).…”

Section: Tmrna Unfoldingmentioning

confidence: 66%

Transfer-messenger RNA unfolds as it transits the ribosome

Zwieb

2005

RNA

“…By analogy with bacterial (4), yeast (23), insect (10), and viral (81) systems, we had originally hypothesized that reliance on a restricted set of nonpreferred codons for the synthesis of capsid region polypeptides might cause a local depletion of the pools of minor tRNAs, thus retarding translocation of the ribosome along the viral mRNA. Ribosomal pausing at nonpreferred codons might impair poliovirus protein synthesis and processing in various ways, such as by amino acid misincorporation (55), by increasing the costs of translational proofreading (9), by frameshifting (18), by premature polypeptide chain termination (55), by degradation of the RNA template (23), and by disruption of the proteolytic processing of the polyprotein (56). On the other hand, it has been suggested that the nonrandom locations of nonpreferred codons between structural elements may facilitate proper folding of the nascent capsid proteins of poliovirus (19) and hepatitis A virus (66).…”

Section: Discussionmentioning

confidence: 99%

Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Burns

Shaw

Campagnoli

et al. 2006

J Virol

228

282

We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N C ) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G؉C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an ϳ10-fold range, and virus yields decreased over an ϳ65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced ϳ3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased ϳ18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (ϳ50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.The use of synonymous codons at unequal frequencies, the codon usage bias, is characteristic of all biological systems (26,27). The strength and direction of codon usage bias are related to the genomic GϩC content and the relative abundance of different isoaccepting tRNAs (reviewed in references 1, 16, and 53). Codon usage can affect the efficiency of gene expression. In bacteria (Escherichia coli) (26, 75), yeast (Saccharomyces cerevisiae) (5, 27), plants (Arabidopsis thaliana) (12), nematodes (Caenorhabditis elegans) (16), and insects (Drosophila melanogaster) (50), the most highly expressed genes use codons matched to the most abundant tRNAs (2). In contrast, in humans and other vertebrates, codon usage bias is much more strongly correlated with the GϩC content of the isochore where the gene is located (51, 71) than with the breadth or level of gene expression (16) or the number of corresponding tRNA genes (28, 30). Despite the weak correlation between codon usage and the levels of gene expression in mammalian cells (16,71), imbalances between codon usage and tRNA abundance can sharply reduce the levels of gene expression.Optimization of codon composition is frequently required for the efficient expression of genes in heterologous host systems (3,31,67,76). For example, the expression of human immunodeficiency virus type 1 ...

Journal of Biological Chemistry

“…Knowing this potential, it is perhaps not surprising that Shine-Dalgarno sequences can also influence landing site selection by bypassing ribosomes because they linearly scan mRNA in the absence of special mRNA signals (4). Nevertheless, it is new, and its implications merit consideration.…”

Section: Discussionmentioning

confidence: 99%

“…Long range translational bypassing in response to A-site codons whose aminoacyl-tRNAs are limiting has been demonstrated (2)(3)(4). In these cases, the peptidyl-tRNA dissociates from the ribosomal P-site codon, scans the coding gap, and re-pairs to mRNA at complementary triplets.…”

mentioning

confidence: 99%

Factors That Influence Selection of Coding Resumption Sites in Translational Bypassing

Herr

Wills

Nelson

et al. 2004

This study investigates bypassing initiated from codons immediately 5 of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3 codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized. These results have implications for re-pairing in ribosomal frameshifting. Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed. The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.Standard genetic decoding is dependent on the formation and maintenance of cognate codon:anticodon pairing. Once established in the ribosomal A-site, this interaction continues through the ribosomal P-site. However, at specific sites in mRNAs ranging from those in retroviruses to Escherichia coli, ribosomes can shift reading frame for gene expression purposes. Most of these specific changes in reading frame involve dissociation of codon:anticodon pairing in the ribosomal P-site and re-pairing at an overlapping codon. Although mechanisms for ensuring the fidelity of pairing in the A-site have been well studied (1), much less is known about the stringency of pairing in the ribosomal P-site. In single nucleotide frameshifting events, the stringency of re-pairing is often difficult to separate from the efficiency of dissociation because the codons share two nucleotides in common. Dissociation and re-pairing, however, are uncoupled in the related phenomenon of translational bypassing. Following initiation of mRNA slippage (or take-off), ribosomes move further downstream, bypassing a block of nucleotides, before coding resumes at the codon 3Ј of the landing site. A careful analysis of landing site selection offers a means to investigate P-site pairing stringency.Long range translational bypassing in response to A-site codons whose aminoacyl-tRNAs are limiting has been demonstrated (2-4). In these cases, the peptidyl-tRNA dissociates from the ribosomal P-site codon, scans the coding gap, and re-pairs to mRNA at complementary triplets. There are also examples of short distance hopping over stop codons