One protein from two open reading frames: mechanism of a 50 nt translational bypass

Translating ribosomes bypass a 50 nt coding gap in order to fuse the information found in the two open reading frames (ORFs) of bacteriophage T4 gene 60. This study investigates the underlying mechanism by focusing on the competition between initiation of bypassing and termination at the end of the first ORF. While nearly all ribosomes initiate bypassing, no more than 50% resume translation in the second ORF. Two previously described cis-acting stimulatory signals are critical for favoring initiation of bypassing over termination. Genetic analysis of these signals supports a working model in which the first (a stem-loop structure at the junction between the first ORF and the coding gap) interferes with decoding in the A-site, and the second (a stretch of amino acids in the nascent peptide encoded by the first ORF) destabilizes peptidyl-tRNA-mRNA pairing.     

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.     

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.     

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.     

Sequence of the short unique region, short repeats, and part of the long repeats of human cytomegalovirus

We have determined the DNA sequence (46 kilobases) of the short unique region, the short repeat, and part of the long repeat of human cytomegalovirus strain AD169. Analysis of the sequence has revealed at least 38 possible regions that may code for protein. Many of these open reading frames show homology to each other, and five groups of homologous reading frames are identified. Half of the predicted translation products appear to be membrane proteins, and fall into two distinct classes; those that have potential signal and anchor sequences, and those that have seven potential membrane-spanning regions and appear to be integral membrane proteins. A number of the former class contain sites for N-linked glycosylation and may therefore be glycoproteins. None of the 38 open reading frames shows homology to other known herpesvirus proteins.     

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.     

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.     

Spontaneous ribosome bypassing in growing cells

Translating ribosomes can pass through a stretch of messenger RNA without translating and resume protein chain elongation after the bypassed region. We previously investigated the stimulation of bypassing when the codon in the ribosome [corrected] A-site called for an aminoacyl-tRNA species in short supply. Here, we investigate bypassing in unstarved, growing cells. A collection of lacZ bypass reporters was constructed with nearly all the sense codons as the "takeoff site", each with its matched landing site 16 nucleotides downstream in the beta-galactosidase reading frame. Beta-galactosidase [corrected] synthesis in unstarved cells carrying these reporters was found to vary over a large range. The takeoff sites UUU and AGG yielded unusually high enzyme activities, sufficient for protein sequence analysis; in these cases, sequencing (by Edman degradation or by mass spectrometry) confirmed that the synthesis of lacZ protein occurred through the 16 nt bypass from takeoff to landing site. Thus, bypassing occurs spontaneously under normal conditions, and is not limited to the pathology of amino acid starvation. Indirect evidence suggests that most of the lower enzyme activities of the rest of the collection also reflects bypassing. Another collection of reporters was made with [corrected] various triplets in the A-site [corrected] the codon immediately following a UUC [corrected] takeoff triplet. Spontaneous bypassing in representatives of this collection varied roughly inversely with the abundance of the tRNA encoded at the A-site. For two A-site codons tested, introduction of additional copies of the relevant tRNA gene on a second plasmid reduced spontaneous bypassing. We conclude that any pause with the A-site empty stimulates bypassing. From the P-site and A-site effects on bypassing, we estimated the average frequency of ribosome takeoff; from this, we calculate that the probability that a ribosome will succeed in translating the entire lacZ coding sequence is about 0.73, in agreement with earlier, independent estimates.     

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.     

The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site

Several retroviruses have recently been shown to promote translation of their gag gene products by internal ribosome entry. In this report, we show that mRNAs containing the human immunodeficiency virus type 1 (HIV-1) gag open reading frame (ORF) exhibit internal ribosome entry site (IRES) activity that can promote translational initiation of Pr55(gag). Remarkably, this IRES activity is driven by sequences within the gag ORF itself and is not dependent on the native gag 5'-untranslated region (UTR). This cap-independent mechanism for Pr55(gag) translation may help explain the high levels of translation of this protein in the face of major RNA structural barriers to scanning ribosomes found in the gag 5' UTR. The gag IRES activity described here also drives translation of a novel 40-kDa Gag isoform through translational initiation at an internal AUG codon found near the amino terminus of the Pr55(gag) capsid domain. Our findings suggest that this low-abundance Gag isoform may be important for wild-type replication of HIV-1 in cultured cells. The activities of the HIV-1 gag IRES may be an important feature of the HIV-1 life cycle and could serve as a novel target for antiretroviral therapeutic strategies.     

Effects of elongation on the translation of a reovirus bicistronic mRNA

The S1 species of reovirus mRNA contains two overlapping open reading frames. Both are utilized in either virus-infected or S1 DNA transfected mammalian cells, resulting in two different polypeptides from a single mRNA. Consistent with ribosome scanning, expression of the downstream reading frame was increased by sequence changes that diminished the consensus around the upstream initiator site. However, the upstream product was not decreased by the same changes, suggesting that its synthesis is rate-limited at elongation. The results suggest a model for regulating bicistronic mRNA translation in which the ribosomes in one reading frame interfere with the movement of ribosomes in the other frame due to different rates of elongation.     

Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap.

The major late 16S mRNA species of simian virus 40 encodes both a 61-amino-acid protein, LP1, and the major virion protein, VP1. Although the initiation signal GCCAUGG is usually utilized at high efficiency, at least one-third of 40S ribosomal subunits bypass it when it is present on the major 16S mRNA of simian virus 40 (S. A. Sedman, P. J. Good, and J. E. Mertz, J. Virol. 63:3884-3893, 1989). The LP1 translation initiation codon is situated 10 bases from the 5' end of this mRNA. To determine whether the short length of the untranslated leader of this mRNA affects the efficiency of translation initiation at the LP1 initiation signal, monkey cells were transfected with plasmids which encode major late 16S-like mRNAs with 5' untranslated regions (UTRs) of 6 or 44 bases. Decreasing the length of the 5' UTR from 44 to 6 bases resulted in a 30% decrease in translation initiation at the LP1 AUG and a threefold increase in synthesis of VP1. When the VP1 open reading frame was replaced with the chloramphenicol acetyltransferase open reading frame, the reduction in 5' UTR length resulted in a 70% decrease in translation initiation at the LP1 AUG and a 30% increase in chloramphenicol acetyltransferase synthesis. Therefore, ribosomes bypass an AUG codon more efficiently when it is located very close to the 5' end of the mRNA.     

Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo.

Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames. We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli. A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA. Only the monomeric forms of GFP produced from circular mRNA are fluorescent. Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA. This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.     

Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.     

Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes

The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and unannotated translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes.     

Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection.

In order to assign specific functions to individual gene products encoded by adenovirus type 5 early region 4 (E4), we have constructed and analyzed a set of mutant viruses that express individual E4 open reading frames or combinations of open reading frames. The results of these analyses demonstrate that the gene products of E4 open reading frames 3 and 6 have redundant effects in viral lytic infection. These E4 products independently augment viral DNA replication, viral late protein synthesis, the shutoff of host cell protein synthesis, and the production of infectious virus. The product of open reading frame 6 is more efficient in the regulation of these processes than is the product of open reading frame 3. The regulation of viral DNA replication and the control of viral and cellular protein synthesis appear to be separable functions associated with both E4 gene products. The role of early region 4 in adeno-associated virus helper function, however, is mediated only by the product of open reading frame 6. Finally, we demonstrate that E4 mutant viruses display a multiplicity-leakiness phenotype which is consistent with the regulatory role that this region plays in viral infection.     

Viral alteration of cellular translational machinery increases defective ribosomal products

Here we show that cells expressing genes inserted into Semliki Forest virus (SFV) vectors generate a large fraction of defective ribosomal products (DRiPs) due to frequent initiation on downstream Met residues. In monopolizing the host cell translational machinery, SFV reduces levels of translation eukaryotic initiation factor 4E (eIF4E), diminishes phosphorylation of ribosome subunit S6, and phosphorylates translation initiation factor eIF2alpha. We show that the last event is required for SFV mistranslation of inserted genes. Downstream initiation is suppressed by fusing inserted genes with the open reading frame encoding the SFV capsid, demonstrating that one function of the capsid element is to enable ribosomes to initiate translation in the proper location. These results show that in modifying translation, viral vectors can unpredictably increase the generation of truncated polypeptides and thereby the DRiP fraction of inserted gene products, which can potentially affect their yield, therapeutic efficacy, and immunogenicity.