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1.
mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot. 相似文献
2.
A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting 总被引:2,自引:0,他引:2
Programmed ribosomal frameshifting (PRF) is a process by which ribosomes produce two different polypeptides from the same mRNA. In this study, we propose three different kinetic models of +1 PRF, incorporating the effects of the ribosomal E-, P- and A-sites toward promoting efficient +1 frameshifting in Escherichia coli. Specifically, the timing of E-site tRNA dissociation is discussed within the context of the kinetic proofreading mechanism of aminoacylated tRNA (aa-tRNA) selection. Mathematical modeling using previously determined kinetic rate constants reveals that destabilization of deacylated tRNA in the E-site, rearrangement of peptidyl-tRNA in the P-site, and availability of cognate aa-tRNA corresponding to the A-site act synergistically to promote efficient +1 PRF. The effect of E-site codon:anticodon interactions on +1 PRF was also experimentally examined with a dual fluorescence reporter construct. The combination of predictive modeling and empirical testing allowed the rate constant for P-site tRNA slippage (ks) to be estimated as ks ≈1.9 s−1 for the release factor 2 (RF2) frameshifting sequence. These analyses suggest that P-site tRNA slippage is the driving force for +1 ribosomal frameshifting while the presence of a ‘hungry codon’ in the A-site and destabilization in the E-site further enhance +1 PRF in E. coli. 相似文献
3.
Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2 总被引:3,自引:1,他引:2
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Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication. 相似文献
4.
Antisense-induced ribosomal frameshifting 总被引:1,自引:0,他引:1
Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels. 相似文献
5.
−1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1–PK4 (∼97% contain PK1, PK3, and PK4), covering ∼72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1–PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of −1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent −1 PRF. 相似文献
6.
Howard Gamper Yujia Mao Isao Masuda Henri McGuigan Gregor Blaha Yuhong Wang Shoujun Xu Ya-Ming Hou 《Nucleic acids research》2021,49(17):10046
Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with –1 frameshifting. 相似文献
7.
In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3′-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt window along a gene sequence according to different effects of the stimulatory signals, and ribosome E-, P- and A-site interactions. FSscan successfully identified the +1 PRF site in prfB and predicted yehP, pepP, nuoE and cheA as +1 frameshift candidates in the E. coli genome. Empirical results demonstrated that potential +1 frameshift sequences identified promoted significant levels of +1 frameshifting in vivo. Mass spectrometry analysis confirmed the presence of the frameshifted proteins expressed from a yehP-egfp fusion construct. FSscan allows a genome-wide and systematic search for +1 frameshift sites in E. coli. The results have implications for bioinformatic identification of novel frameshift proteins, ribosomal frameshifting, coding sequence detection and the application of mass spectrometry on studying frameshift proteins. 相似文献
8.
Daniella Ishimaru Ewan P. Plant Amy C. Sims Boyd L. Yount Jr Braden M. Roth Nadukkudy V. Eldho Gabriela C. Pérez-Alvarado David W. Armbruster Ralph S. Baric Jonathan D. Dinman Deborah R. Taylor Mirko Hennig 《Nucleic acids research》2013,41(4):2594-2608
Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis. 相似文献
9.
Michel de?Messieres Jen-Chien Chang Ashton?Trey Belew Arturas Meskauskas Jonathan?D. Dinman Arthur La?Porta 《Biophysical journal》2014,106(1):244-252
Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a −1 programmed ribosomal frameshift (−1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human −1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the −1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism. 相似文献
10.
Marie-Hélène Mazauric Jean-Louis Leroy Koen Visscher Satoko Yoshizawa Dominique Fourmy 《RNA (New York, N.Y.)》2009,15(9):1775-1786
Many viruses regulate translation of polycistronic mRNA using a −1 ribosomal frameshift induced by an RNA pseudoknot. When the ribosome encounters the pseudoknot barrier that resists unraveling, transient mRNA–tRNA dissociation at the decoding site, results in a shift of the reading frame. The eukaryotic frameshifting pseudoknot from the beet western yellow virus (BWYV) has been well characterized, both structurally and functionally. Here, we show that in order to obtain eukaryotic levels of frameshifting efficiencies using prokaryotic Escherichia coli ribosomes, which depend upon the structural integrity of the BWYV pseudoknot, it is necessary to shorten the mRNA spacer between the slippery sequence and the pseudoknot by 1 or 2 nucleotides (nt). Shortening of the spacer is likely to re-establish tension and/or ribosomal contacts that were otherwise lost with the smaller E. coli ribosomes. Chemical probing experiments for frameshifting and nonframeshifting BWYV constructs were performed to investigate the structural integrity of the pseudoknot confined locally at the mRNA entry site. These data, obtained in the pretranslocation state, show a compact overall pseudoknot structure, with changes in the conformation of nucleotides (i.e., increase in reactivity to chemical probes) that are first “hit” by the ribosomal helicase center. Interestingly, with the 1-nt shortened spacer, this increase of reactivity extends to a downstream nucleotide in the first base pair (bp) of stem 1, consistent with melting of this base pair. Thus, the 3 bp that will unfold upon translocation are different in both constructs with likely consequences on unfolding kinetics. 相似文献
11.
Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate −1 programmed ribosomal frameshifting (PRF), suggesting −1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the −1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent −1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of −1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent −1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA–ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific −1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application. 相似文献
12.
Pei-Yu Liao Yong Seok Choi Jonathan D. Dinman Kelvin H. Lee 《Nucleic acids research》2011,39(1):300-312
Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed −1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe −1 PRF. The model reveals three kinetic pathways to −1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating −1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to −1 PRF and the ratio between two types of frameshift products were evaluated. A dual fluorescence reporter was employed in Escherichia coli to empirically test the model. Additionally, the study applied a novel mass spectrometry approach to quantify the ratios of the two frameshift products. A more detailed understanding of the mechanisms underlying −1 PRF may provide insight into developing antiviral therapeutics. 相似文献
13.
Guan Wang Yang Yang Xiaolan Huang 《Journal of biomolecular structure & dynamics》2013,31(7):1547-1557
?1 programmed ribosomal frameshifting (PRF) in viruses is often stimulated by a pseudoknot downstream from the slippery sequence. At the PRF junction of HIV-1, transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV), we identified potential double pseudoknots in either a tandem mode or embedded mode. In viruses with tandem pseudoknots (5′PK & 3′PK), the slippery sequence is encompassed in the 5′PK. The ribosome needs to unwind the 5′PK to get to the slippery sequence. In HIV-1, the 3′PK and several alternative structures are mutually exclusive. Disruption of the tandem pseudoknots may enable one of the alternative structures to form as the effective frameshift stimulator. In TGEV/BFV/FMV, the 3′PK is a conventional frameshift stimulator. In all cases, the tandem pseudoknots may slow down the ribosome before it reaches the conventional PRF signals. In EAV, a compact pseudoknot is embedded within loop2 of the otherwise conventional frameshift-stimulating pseudoknot. All double pseudoknots have the potential to stack their stems coaxially. We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded modes are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure, and avoid an ultra-stable single pseudoknot which may become a ribosomal roadblock. Our results significantly expand the repertoire of RNA structures and dynamics that may potentially involve in ?1 PRF regulation. 相似文献
14.
Yoshifumi Kobayashi Jianling Zhuang Stuart Peltz Joseph Dougherty 《The Journal of biological chemistry》2010,285(26):19776-19784
Programmed −1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses, including human immunodeficiency virus type 1 (HIV-1), to produce multiple proteins from a single mRNA. −1 PRF induces a subset of elongating ribosomes to shift their translational reading frame by 1 base in the 5′ direction. The appropriate ratio of Gag to Gag-Pol synthesis is tightly regulated by the PRF signal which promotes ribosomes to shift frame, and even small changes in PRF efficiency, either up or down, have significant inhibitory effects upon virus production, making PRF essential for HIV-1 replication. Although little has been reported about the cellular factors that modulate HIV-1 PRF, the cis-acting elements regulating PRF have been extensively investigated, and the PRF signal of HIV-1 was shown to include a slippery site and frameshift stimulatory signal. Recently, a genome-wide screen performed to identify cellular factors that affect HIV-1 replication demonstrated that down-regulation of eukaryotic release factor 1 (eRF1) inhibited HIV-1 replication. Because of the eRF1 role in translation, we hypothesized that eRF1 is important for HIV-1 PRF. Using a dual luciferase reporter system harboring a HIV-1 PRF signal, results showed that depletion or inhibition of eRF1 enhanced PRF in yeast, rabbit reticulocyte lysates, and mammalian cells. Consistent with the eRF1 role in modulating HIV PRF, depleting eRF1 increased the Gag-Pol to Gag ratio in cells infected with replication-competent virus. The increase in PRF was independent of a proximal termination codon and did not result from increased ribosomal pausing at the slippery site. This is the first time that a cellular factor has been identified which can promote HIV-1 PRF and highlights HIV-1 PRF as essential for replication and an important but under exploited antiviral drug target. 相似文献
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16.
Programmed -1 ribosomal frameshifting (-1 PRF) is used by many positive-strand RNA viruses for translation of required products. Despite extensive studies, it remains unresolved how cis-elements just downstream of the recoding site promote a precise level of frameshifting. The Umbravirus Pea enation mosaic virus RNA2 expresses its RNA polymerase by -1 PRF of the 5′-proximal ORF (p33). Three hairpins located in the vicinity of the recoding site are phylogenetically conserved among Umbraviruses. The central Recoding Stimulatory Element (RSE), located downstream of the p33 termination codon, is a large hairpin with two asymmetric internal loops. Mutational analyses revealed that sequences throughout the RSE and the RSE lower stem (LS) structure are important for frameshifting. SHAPE probing of mutants indicated the presence of higher order structure, and sequences in the LS may also adapt an alternative conformation. Long-distance pairing between the RSE and a 3′ terminal hairpin was less critical when the LS structure was stabilized. A basal level of frameshifting occurring in the absence of the RSE increases to 72% of wild-type when a hairpin upstream of the slippery site is also deleted. These results suggest that suppression of frameshifting may be needed in the absence of an active RSE conformation. 相似文献
17.
18.
The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae
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Nilgun E. Tumer Bijal A. Parikh Ping Li Jonathan D. Dinman 《Journal of virology》1998,72(2):1036-1042
Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M1-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity. 相似文献
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20.
Studies of programmed -1 ribosomal frameshifting (-1 PRF) have been approached over the past two decades by many different laboratories using a diverse array of virus-derived frameshift signals in translational assay systems derived from a variety of sources. Though it is generally acknowledged that both absolute and relative -1 PRF efficiency can vary in an assay system-dependent manner, no methodical study of this phenomenon has been undertaken. To address this issue, a series of slippery site mutants of the SARS-associated coronavirus frameshift signal were systematically assayed in four different eukaryotic translational systems. HIV-1 promoted frameshifting was also compared between Escherichia coli and a human T-cell line expression systems. The results of these analyses highlight different aspects of each system, suggesting in general that (1) differences can be due to the assay systems themselves; (2) phylogenetic differences in ribosome structure can affect frameshifting efficiency; and (3) care must be taken to employ the closest phylogenetic match between a specific -1 PRF signal and the choice of translational assay system. 相似文献