Maintenance of the correct open reading frame by the ribosome

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon–anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.     

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.     

Structural determinants of an internal ribosome entry site that direct translational reading frame selection

The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome.     

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5'-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3'-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5'- and 3'-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.     

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

Open reading frame analysis by selective PCR-mediated deletion mutagenesis

Recently, we demonstrated that a nested set of DNA fragments can be obtained by using one specific primer and one semirandom primer in a polymerase chain reaction (PCR). We now describe a strategy for selective deletion mutagenesis that is based on this observation. The gene of interest is cloned as a fusion construct with a selectable marker in a small vector, allowing for PCR amplification of the entire recombinant plasmid. The specific primer is complementary to the vector sequence beyond the gene of interest and is oriented downstream. The 3′ end of the semirandom primer is complementary to a triplet (GAT) that is scattered over the entire open reading frame (ORF). It is shown by nucleotide sequence analysis that deletion mutants result exclusively from annealing of the semirandom primer at different GAT triplets. PCR products resulting fro from annealing to GAT triplets elsewhere in the plasmid are counterselected by the need for replication functions and for the expression of the selectable marker. This technique is demonstrated on the Saccharomyces cerevisiae ORF YCL56C.     

Posttranscriptional regulation by the upstream open reading frame of the phosphoethanolamine N-methyltransferase gene

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5' untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5' UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the beta-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream beta-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.     

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.     

Pulling the ribosome out of frame by +1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA.

Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation. A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tRNA occupying the P site during the pause. The special nature of the peptidyl-tRNA reflects its ability to slip +1 on the mRNA or to facilitate binding of an incoming aminoacyl-tRNA out of frame in the A site. This second mechanism suggested that in some cases the first +1 frame tRNA could have an active role in frameshifting. We found that overproducing this tRNA can drive frameshifting, surprisingly regardless of whether frameshifting occurs by peptidyl-tRNA slippage or out-of-frame binding of aminoacyl-tRNA. This finding suggests that in both cases, the shift in reading frame occurs coincident with formation of a cognate codon-anticodon interaction in the shifted frame.     

Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the “Hirsh” mutation). We used the Escherichia coli RF2 programmed frameshift site to monitor frame maintenance. Most anticodon loop mutations increase frameshifting, possibly by decreasing codon:anticodon stability. However, it is likely that the A site is more sensitive to anticodon loop structure than is the E site. Unexpectedly, the Hirsh mutation also increases frameshifting from the E site. Other work shows that mutation may increase the ability of tRNA to react in the A site, possibly by facilitating conformational changes required for aminoacyl-tRNA selection. We suggest that this property may decrease its ability to bind to the E site. Finally, the absence of the ms²io⁶A nucleoside modifications at A37 does not decrease the ability of tRNA to help hold the reading frame from the E site. This was also unexpected because the absence of these modifications affects translational properties of tRNA in A and P sites. The absence of a negative effect in the E site further highlights the differences among the substrate requirements of the ribosomal coding sites.     

Characterization of a triplex DNA-binding protein encoded by an alternative reading frame of loricrin.

In an attempt to identify genes encoding triple-helical DNA-binding proteins, we performed South-Western screening of a human keratinocyte cDNA expression library using a purine (Pu)-rich triplex DNA probe. We isolated two independent clones containing part of the loricrin gene. Both were translated with a different reading frame than that of the loricrin protein, the major component of the cell envelope during normal keratinocyte cornification. The affinity of the encoded polypeptide for Pu-triplex DNA was confirmed by electrophoretic mobility shift assays using a bacterially expressed N-terminal loricrin deletion fused with the maltose-binding protein (MBP-LOR3ARF). Interactions between Pu-triplex DNA and MBP-LOR3ARF are characterized by a distribution of four increasingly slower mobility complexes, suggesting that multiple MBP-LOR3ARF molecules can recognize a single triplex. Binding was also observed between MBP-LOR3ARF and a pyrimidine-motif triplex DNA, although at lower affinity than Pu-triplex DNA. No apparent binding was observed between MBP-LOR3ARF and double-stranded DNA, suggesting that MBP-LOR3ARF is a bona fide Pu-triplex binding protein. Finally, purified specific rabbit antibodies against LORARF detected four human proteins with apparent molecular masses of 210, 110, 68, and 66 kDa on Western blot analysis. The 210-, 110-, and 68-kDa proteins also showed specific Pu-triplex DNA binding in a South-Western experiment, suggesting that LORARF shares common domains with other human Pu-triplex DNA-binding proteins.     

Properties of the protein encoded by the UL32 open reading frame of herpes simplex virus 1.

The functions previously assigned to the essential herpes simplex virus 1 UL32 protein were in cleavage and/or packaging of viral DNA and in maturation and/or translocation of viral glycoproteins to the plasma membrane. The amino acid sequence predicts N-linked glycosylation sites and sequences conserved in aspartyl proteases and in zinc-binding proteins. We report the following. (i) The 596-amino-acid UL32 protein accumulated predominantly in the cytoplasm of infected cells but was not metabolically labeled with glucosamine and did not band with membranes containing a known glycoprotein in flotation sucrose density gradients. The UL32 protein does not, therefore, have the properties of an intrinsic membrane protein. (ii) Experiments designed to demonstrate aspartyl protease activity in a phage display system failed to reveal proteolytic activity. Moreover, substitution of Asp-110 with Gly in the sequence Asp-Thr-Gly, the hallmark of aspartyl proteases, had no effect on viral replication in Vero and SK-N-SH cell lines or in human foreskin fibroblasts. Therefore, if the UL32 protein functions as a protease, this function is not required in cells in culture. (iii) Both the native UL32 protein and a histidine-tagged UL32 protein made in recombinant baculovirus-infected insect cells bound zinc. The consensus sequence is conserved in the UL32 homologs from varicella-zoster virus and equine herpesvirus 1. UL32 protein is therefore a cysteine-rich, zinc-binding essential cytoplasmic protein whose function is not yet clear.