Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae

Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin.     

The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene. mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.     

Ribosomal Protein L3 Mutants Alter Translational Fidelity and Promote Rapid Loss of the Yeast Killer Virus

Programmed −1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation. We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed −1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast. Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome’s peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss. Mutations in the RPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed −1 ribosomal frameshift efficiencies and loss of the M₁ killer virus of yeast. The mak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p. Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Δ) exerts a trans-dominant effect on programmed −1 ribosomal frameshifting and killer virus maintenance. Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed −1 ribosomal frameshifting.     

Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis

There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed -1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5' (7)mGppp cap structure and 3' poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap(+)/poly(A)(+) and cap(-)/poly(A)(-) mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.     

Mof4-1 is an allele of the UPF1/IFS2 gene which affects both mRNA turnover and -1 ribosomal frameshifting efficiency.

The mof4-1 (maintenance of frame) allele in the yeast Saccharomyces cerevisiae was isolated as a chromosomal mutation that increased the efficiency of -1 ribosomal frameshifting at the L-A virus frameshift site and caused loss of M1, the satellite virus of L-A. Here, we demonstrate that strains harboring the mof4-1 allele inactivated the nonsense-mediated mRNA decay pathway. The MOF4 gene was shown to be allelic to UPF1, a gene whose product is involved in the nonsense-mediated mRNA decay pathway. Although cells harboring the mof4-1 allele of the UPF1 gene lose the M1 virus, mutations in other UPF genes involved in nonsense-mediated mRNA decay maintain the M1 virus. The mof4-1 strain is more sensitive to the aminoglycoside antibiotic paromomycin than a upf1 delta strain, and frameshifting efficiency increases in a mof4-1 strain grown in the presence of this drug. Further, the ifs1 and ifs2 alleles previously identified as mutations that enhance frameshifting were shown to be allelic to the UPF2 and UPF1 genes, respectively, and both ifs strains maintained M1. These results indicate that mof4-1 is a unique allele of the UPF1 gene and that the gene product of the mof4-1 allele affects both -1 ribosomal frameshifting and mRNA turnover.     

The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

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.     

Translational Maintenance of Frame: Mutants of Saccharomyces Cerevisiae with Altered -1 Ribosomal Frameshifting Efficiences

A special site on the (+) strand of the L-A dsRNA virus induces about 2% of ribosomes translating the gag open reading frame to execute a -1 frameshift and thus produce the viral gag-pol fusion protein. Using constructs in which a -1 ribosomal frameshift at this site was necessary for expression of lacZ we isolated chromosomal mutants in which the efficiency of frameshifting was increased. These mutants comprise eight genes, named mof (maintenance of frame). The mof1-1, mof2-1, mof4-1, mof5-1 and mof6-1 strains cannot maintain M(1) dsRNA at 30°, but, paradoxically, do not lose L-A. The mof2-1, mof5-1 and mof6-1 strains are temperature sensitive for growth at 37°, and all three show striking cell cycle phenotypes. The mof2-1 strains arrest with mother and daughter cells almost equal in size, mof5-1 arrests with multiple buds and mof6-1 arrests as single large unbudded cells. mof2-1 and mof5-1 strains are also Pet(-). The mof mutations show differential effects on various frameshifting signals.     

Decreased peptidyltransferase activity correlates with increased programmed -1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae

Increased efficiencies of programmed -1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed -1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed -1 ribosomal frameshift signals.     

Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity.

The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus.     

5 S Rrna Is Involved in Fidelity of Translational Reading Frame

Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frame-shifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof(-) phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof(-) phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.     

Kinetics of ribosomal pausing during programmed -1 translational frameshifting

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.     

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.     

Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.     

Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus

Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.     

Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.     

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.     

E. coli ribosomes re-phase on retroviral frameshift signals at rates ranging from 2 to 50 percent

Many retroviruses express gag-pol or gag-pro-pol polypeptides by coupling their translation from overlapping reading frames with -1 ribosomal frameshifts. Here, we show that the well-known ribosomal frameshift signals found in retroviral mRNA will provoke Escherichia coli ribosomes to shift frame in the same manner as their eukaryotic counterparts. Ribosomes of E. coli respond in vivo to both the tandem slippery codons present at the retroviral frameshift site and the 3' flanking sequence. Slight alteration of the mouse mammary tumor virus gag-pro frameshift site from A-AAA-AAC to A-AAA-AAG boosts the level of frameshifting in E. coli to over 50%. This suggests that A-AAA-AAG, and its slippery relatives, may be utilized by E. coli genes as sites of high-level ribosomal frameshifting. This observed conservation of response to retroviral frameshift signals affords new avenues to dissect the mechanism of ribosomal frameshifting evoked by these mRNA sequences.     

mRNA pseudoknot structures can act as ribosomal roadblocks

Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses.     

Footprinting analysis of BWYV pseudoknot–ribosome complexes

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.