Computational identification of putative programmed translational frameshift sites

MOTIVATION: In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS: Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY: The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION: Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis     

Conserved translational frameshift in dsDNA bacteriophage tail assembly genes

A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.     

RNA-binding protein-mediated translational repression of transgene expression in plants

We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants. RNA sequences called translational operators were inserted at a cap-proximal position in the 5-UTR of mRNAs of two reporter genes, gusor aroA:CP4. Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed. The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells. Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent. A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal. Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.these authors contributed equally to this workthese authors contributed equally to this work     

Control of translational repression by protein-protein interactions.

The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.     

General translational repression by activators of mRNA decapping

Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit translation and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing non-translating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation. Strains lacking both Dhh1p and Pat1p show strong defects in mRNA decapping and P body formation and are blocked in translational repression. Contrastingly, overexpression of Dhh1p or Pat1p causes translational repression, P body formation, and arrests cell growth. Dhh1p, and its human homolog, RCK/p54, repress translation in vitro, and Dhh1p function is bypassed in vivo by inhibition of translational initiation. These results identify a broadly acting mechanism of translational repression that targets mRNAs for decapping and functions in translational control. We propose this mechanism is competitively balanced with translation, and shifting this balance is an important basis of translational control.     

Differential response to frameshift signals in eukaryotic and prokaryotic translational systems.

The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E. coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E. coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E. coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leafroll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent.     

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.     

Programmed translational frameshift in the bacteriophage P2 FETUD tail gene operon

The major structural components of the P2 contractile tail are encoded in the FETUD tail gene operon. The sequences of genes F(I) and F(II), encoding the major tail sheath and tail tube proteins, have been reported previously (L. M. Temple, S. L. Forsburg, R. Calendar, and G. E. Christie, Virology 181:353-358, 1991). Sequence analysis of the remainder of this operon and the locations of amber mutations Eam30, Tam5, Tam64, Tam215, Uam25, Uam77, Uam92, and Dam6 and missense mutation Ets55 identified the coding regions for genes E, T, U, and D, completing the sequence determination of the P2 genome. Inspection of the DNA sequence revealed a new open reading frame overlapping the end of the essential tail gene E. Lack of an apparent translation initiation site and identification of a putative sequence for a programmed translational frameshift within the E gene suggested that this new reading frame (E') might be translated as an extension of gene E, following a -1 translational frameshift. Complementation analysis demonstrated that E' was essential for P2 lytic growth. Analysis of fusion polypeptides verified that this reading frame was translated as a -1 frameshift extension of gpE, with a frequency of approximately 10%. The arrangement of these two genes within the tail gene cluster of phage P2 and their coupling via a translational frameshift appears to be conserved among P2-related phages. This arrangement shows a striking parallel to the organization in the tail gene cluster of phage lambda, despite a lack of amino acid sequence similarity between the tail gene products of these phage families.     

Selective translation of mRNAs at synapses

Synaptic efficacy, a phenomenon that may underlie long-term memory storage, is controlled in part by the regulated translation of mRNAs stored in dendrites. The molecular basis by which specific mRNAs are selected for translation is beginning to emerge and appears to involve at least one mechanism that helps program early metazoan development. Because different neural transmitters elicit different synaptic responses that rely on local protein synthesis, a number of sequence-specific mRNA translational regulatory mechanisms are likely to function in neurons. Such mechanisms may be inferred from those operating in early development and in cognitive disease.     

Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs

Many of the chloroplast mRNAs possess Shine-Dalgarno (SD)-like sequences (typically GGAGG) in the 5'-untranslated regions, but the position is highly variable. Using a homologous in vitro translation system, we assessed the role for translation of SD-like sequences in four tobacco chloroplast mRNAs. The rbcL mRNA has a typical SD-like sequence at a position similar to the conserved position (-12 to -4 with respect to the start codon) observed in E. coli, and this sequence was found to be essential for translation. This was also the case for the atpE mRNA. However, SD-like sequences in the rps12 mRNA and in the petB mRNA is located far from (-44 to -42) and too close to (-5 to -2) the initiation codon, respectively, and these sequences were not essential for translation. These results indicate that functional SD-like sequences are located around 10 nucleotides upstream from the translational start codon. Competition assays confirmed that a functional SD-like sequence interacts with the 3' terminus of chloroplast 16S rRNA.     

Differential translational efficiency of the mRNAs isolated from derepressed and glucose repressed Saccharomyces cerevisiae

Carbon catabolite derepression induced changes in the pool of yeast mRNAs translatable in a protein-synthesizing reticulocyte system. Competition experiments with globin mRNA showed that the mRNA population obtained from derepressed cells possessed a higher translational efficiency than mRNA from repressed cells. The mRNAs that could account for the high translational efficiency of the derepressed mRNA were not detected in cells growing in glucose-rich medium. Analysis of protein synthesis in the presence of 7-methylguanosine 5'-phosphate indicated that the initiation factors recognizing the 5'-terminal structure of capped messengers interacted with lower affinity with the repressed than with some specific derepressed mRNAs.