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1.
The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (ΔL9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ΔL9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest. 相似文献
2.
Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes. 相似文献
3.
Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes. 相似文献
4.
Sharma P Yan F Doronina VA Escuin-Ordinas H Ryan MD Brown JD 《Nucleic acids research》2012,40(7):3143-3151
Expression of viral proteins frequently includes non-canonical decoding events ('recoding') during translation. '2A' oligopeptides drive one such event, termed 'stop-carry on' recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to dictate an unusual stop codon-independent termination of translation at the final Pro codon of 2A. Subsequently, translation 'reinitiates' on the same codon, two individual proteins being generated from one open reading frame. Many 2A peptides have been identified, and they have a conserved C-terminal motif. Little similarity is present in the N-terminal portions of these peptides, which might suggest that these amino acids are not important in the 2A reaction. However, mutagenesis indicates that identity of the amino acid at nearly all positions of a single 2A peptide is important for activity. Each 2A may then represent a specific solution for positioning the conserved C-terminus within the peptidyl-transferase centre to promote recoding. Nascent 2A peptide:ribosome interactions are suggested to alter ribosomal fine structure to discriminate against prolyl-tRNA(Pro) and promote termination in the absence of a stop codon. Such structural modifications may account for our observation that replacement of the final Pro codon of 2A with any stop codon both stalls ribosome processivity and inhibits nascent chain release. 相似文献
5.
A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons. 相似文献
6.
Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression. 相似文献
7.
8.
Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNASec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons. 相似文献
9.
Jesse Donovan 《Journal of molecular biology》2010,400(4):659-361
Selenocysteine (Sec) incorporation is an essential process required for the production of at least 25 human selenoproteins. This unique amino acid is co-translationally incorporated at specific UGA codons that normally serve as termination signals. Recoding from stop to Sec involves a cis-acting Sec insertion sequence element in the 3′ untranslated region of selenoprotein mRNAs as well as Sec insertion sequence binding protein 2, Sec-tRNASec, and the Sec-specific elongation factor, eEFSec. The interplay between recoding and termination at Sec codons has served as a focal point in researching the mechanism of Sec insertion, but the role of translation initiation has not been addressed. In this report, we show that the cricket paralysis virus intergenic internal ribosome entry site is able to support Sec incorporation, thus providing evidence that the canonical functions of translation initiation factors are not required. Additionally, we show that neither a 5′ cap nor a 3′ poly(A) tail enhances Sec incorporation. Interestingly, however, the presence of the internal ribosome entry site significantly decreases Sec incorporation efficiency, suggesting a role for translation initiation in regulating the efficiency of UGA recoding. 相似文献
10.
Mimivirus and Megavirus are the best characterized representatives of an expanding new family of giant viruses infecting Acanthamoeba. Their most distinctive features, megabase-sized genomes carried in particles of size comparable to that of small bacteria, fill the gap between the viral and cellular worlds. These giant viruses are also uniquely equipped with genes coding for central components of the translation apparatus. The presence of those genes, thought to be hallmarks of cellular organisms, revived fundamental interrogations on the evolutionary origin of these viruses and the link they might have with the emergence of eukaryotes. In this work, we focused on the Mimivirus-encoded translation termination factor gene, the detailed primary structure of which was elucidated using computational and experimental approaches. We demonstrated that the translation of this protein proceeds through two internal stop codons via two distinct recoding events: a frameshift and a readthrough, the combined occurrence of which is unique to these viruses. Unexpectedly, the viral gene carries an autoregulatory mechanism exclusively encountered in bacterial termination factors, though the viral sequence is related to the eukaryotic/archaeal class-I release factors. This finding is a hint that the virally-encoded translation functions may not be strictly redundant with the one provided by the host. Lastly, the perplexing occurrence of a bacterial-like regulatory mechanism in a eukaryotic/archaeal homologous gene is yet another oddity brought about by the study of giant viruses. 相似文献
11.
12.
Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b 总被引:11,自引:0,他引:11
Boulant S Becchi M Penin F Lavergne JP 《The Journal of biological chemistry》2003,278(46):45785-45792
13.
A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme. 相似文献
14.
Cobucci-Ponzano B Conte F Benelli D Londei P Flagiello A Monti M Pucci P Rossi M Moracci M 《Nucleic acids research》2006,34(15):4258-4268
The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea. 相似文献
15.
Regulation of protein synthesis at the level of translation termination is a relatively underexplored, but rapidly expanding
field. Recent advances in elucidating the mechanism of translation termination are helping to understand noncanonical events
associated with translation termination. These “recording” events include read-through of stop codons, insertion of unusual
amino acids such as selenocysteine, and production of several polypeptides from one open reading frame. This review summarizes
data on termination-dependent recording events and proposes that there are two types of stop codon-associated sequences optimized
to perform different functions: termination of translation per se or alternative elongation events.
Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 731–741.
The article is published in the original. 相似文献
16.
Recoding refers to the reprogramming of mRNA translation by nonstandard read-out rules. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) technology to investigate the proteome of host-adapted Salmonella serovars, which are characteristic of accumulation of pseudogenes. Interestingly, a few annotated pseudogenes were indeed able to express peptides downstream of the inactivation site, suggesting the occurrence of recoding. Two mechanisms of recoding, namely, programmed frameshifting and codon redefinition, were both found. We believe that the phenomena of recoding are not rare in bacteria. More studies are required for a better understanding of bacterial translation and the implication of pseudogene recoding in Salmonella serovars. 相似文献
17.
《Critical reviews in biochemistry and molecular biology》2013,48(4):257-265
The discovery of the genetic code provided one of the basic foundations of modern molecular biology. Most organisms use the same genetic language, but there are also well-documented variations representing codon reassignments within specific groups of organisms (such as ciliates and yeast) or organelles (such as plastids and mitochondria). In addition, duality in codon function is known in the use of AUG in translation initiation and methionine insertion into internal protein positions as well as in the case of selenocysteine and pyrrolysine insertion (encoded by UGA and UAG, respectively) in competition with translation termination. Ambiguous meaning of CUG in coding for serine and leucine is also known. However, a recent study revealed that codons in any position within the open reading frame can serve a dual function and that a change in codon meaning can be achieved by availability of a specific type of RNA stem-loop structure in the 3′-untranslated region. Thus, duality of codon function is a more widely used feature of the genetic code than previously known, and this observation raises the possibility that additional recoding events and additional novel features have evolved in the genetic code. 相似文献
18.
A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons
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Tony S. Cardno Elizabeth S. Poole Suneeth F. Mathew Ryan Graves Warren P. Tate 《RNA (New York, N.Y.)》2009,15(8):1614-1621
Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z′-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency. 相似文献
19.
A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go)
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Atkins JF Wills NM Loughran G Wu CY Parsawar K Ryan MD Wang CH Nelson CC 《RNA (New York, N.Y.)》2007,13(6):803-810
When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence. This is an example of recoding where 2A promotes unconventional termination after decoding of the glycine codon and continued translation beginning with the 3' adjacent proline codon. 相似文献
20.
SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging. 相似文献