Efficiency of T4 Gene 60 Translational Bypassing

Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene 60 mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene 60 (which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of ~50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.     

Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF-2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination.     

Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing.Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.     

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb³⁺ ions and the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb³⁺ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem–loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.     

Dissecting eukaryotic translation and its control by ribosome density mapping

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5′ and 3′ ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.     

Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency.

Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing.     

Drop-off during ribosome hopping

Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism.     

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.     

A bipartite sequence motif induces translation reinitiation in feline calicivirus RNA

The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.     

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.     

P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation

Programmed ribosomal bypassing occurs in decoding phage T4 gene 60 mRNA. Half the ribosomes bypass a 50 nucleotide gap between codons 46 and 47. Peptidyl-tRNA dissociates from the "take-off" GGA, codon 46, and re-pairs to mRNA at a matched GGA "landing site" codon directly 5' of codon 47 where translation resumes. The system described here allows the contribution of peptidyl-tRNA re-pairing to be measured independently of dissociation. The matched GGA codons have been replaced by 62 other matched codons, giving a wide range of bypassing efficiencies. Codons with G or C in either or both of the first two codon positions yielded high levels of bypassing. The results are compared with those from a complementary study of non-programmed bypassing, where the combined effects of peptidyl-tRNA dissociation and reassociation were measured. The wild-type, GGA, matched codons are the most efficient in their gene 60 context in contrast to the relatively low value in the non-programmed bypassing study.     

5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5′-untranslated regions (5′-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, ~10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.     

Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.     

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.     

The Ayerst Award Lecture 1976. Novel factors in protein biosynthesis.

Comparison of nucleotide sequences surrounding the initiation sites of a number of mRNAs reveals few common features. These may be the presence of in- or out-of-phase nonsense codons and (or) polypurine bases complementary to the 16S RNA of the 30S subunit of ribosomes. Since the bases which precede or follow an initiation site vary in length and composition we have examined whether they play a role as spacers between cistrons or whether they have an active function in the termination and initiation of translation. In vitro we have observed that some sequences 5' terminal to AUG are preferred over others in forming an initiation complex. The same bases have much less effect when present at the 3' terminal end of an AUG codon. When the 5' terminal codon is the termination codon UAA, absolutely no initiation complex can be detected. This suggests that spacing may be needed between a stop and a start codon. Conversely, the hexamer AUGUAA failed to elicit chain termination. This was so in systems that terminated when free UAA was added or when a sense triplet was present between the initiation and termination triplets. These results suggest that ribosomes may recognize the stop triplet. Hence ribosomes may not obey simple A and P site models in the termination reaction.     

Ribosome stalling is responsible for arginine-specific translational attenuation in Neurospora crassa.

The Neurospora crassa arg-2 upstream open reading frame (uORF) plays a role in negative arginine-specific translational regulation. Primer extension inhibition analyses of arg-2 uORF-containing RNA translated in a cell-free system in which arginine-specific regulation was retained revealed "toeprints" corresponding to ribosomes positioned at the uORF initiation and termination codons and at the downstream initiation codon. At high arginine concentrations, the toeprint signal corresponding to ribosomes at the uORF termination codon rapidly increased; a new, broad toeprint that represents additional ribosomes stalled on the uORF appeared 21 to 30 nucleotides upstream of this site; and the toeprint signal corresponding to ribosomes at the downstream initiation codon decreased. These data suggest that arginine increases ribosomal stalling and thereby decreases translation from the downstream initiation codon.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.