RNA sequence determinants of a coupled termination-reinitiation strategy for downstream open reading frame translation in Helminthosporium victoriae virus 190S and other victoriviruses (Family Totiviridae)

The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.     

The actin gene in yeast Saccharomyces cerevisiae: 5'' and 3'' end mapping, flanking and putative regulatory sequences

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.     

Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

5'-proximal hot spot for an inducible positive-to-negative-strand template switch by coronavirus RNA-dependent RNA polymerase

Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.     

The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase

The DNA sequence of the gene for the fermentative yeast alcohol dehydrogenase has been determined. The structural gene contains no introns. The amino acid sequence of the protein as determined from the nucleotide sequence disagrees with the published alcohol dehydrogenase isozyme I (ADH-I) sequence for 5 of the 347 amino acid residues. At least one, and perhaps as many as four, of these differences is probably due to ADH-I protein heterogeneity in different yeast strains and not to sequencing errors. S1 nuclease was used to map the 5' and 3' ends of the ADH-I mRNA. There are two discrete, mature 5' ends of the mRNA, mapping 27 and 37 nucleotides upstream of the translation initiating ATG. These two equally prevalent termini are 101 and 91 nucleotides, respectively, downstream from a TATAAA sequence. Analysis of the 3' end of ADH-I mRNA disclosed two minor ends upstream of the major poly(A) addition site. These three ends map 24, 67, and 83 nucleotides, respectively, downstream from the translation-terminating TAA triplet. The sequence AA-TAAG is found 28 to 34 nucleotides upstream of each ADH-I mRNA poly(A) addition site. Sequence comparisons of these three 3' ends with those for four other yeast mRNAs yielded a 13-nucleotide consensus sequence to which TAAATAAGA is central. All of the known yeast poly(A) addition sites map at or near the A residue of a CTA site 25 to 40 nucleotides downstream from this consensus octamer.     

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.     

NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein

The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.