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Results for “Codon, Initiator”

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PubMed1990

Translation of the Saccharomyces cerevisiae tcm1 gene in the absence of a 5'-untranslated leader.

The role of eukaryotic 5'-untranslated messenger RNA leaders is not entirely clear, since they share little sequence similarity among each other. The importance of the leader in determining the efficiency of translation initiation was addressed here by examining the polyribosome distribution of several leader-deletion alleles of the yeast tcm1 gene (coding for ribosomal protein L3). Shortening of this 22-nucleotide leader, or complete removal of it (the first nucleotide of the mRNA becoming the A of the translation initiation codon AUG) permitted translation, albeit reduced. Further deletion of as few as the first two nucleotides of the initiation codon leads to a substantial reduction in ribosome loading, which is compatible with inefficient initiation at the next downstream, out-of-frame, AUG triplet. A second measure of translation initiation was obtained by assaying qualitatively for the production of biologically active L3 protein using growth-resistance to trichodermin. This experiment indicates that ribosomes can recognize the correct initiation codon even in the complete absence of a leader. We conclude that the 5'-untranslated leader of the yeast tcm1 gene is not essential for accurate translation initiation, but enhances its efficiency.

Base Sequence↗

PubMed1987

High-frequency deletional rearrangement of immunoglobulin kappa gene segments introduced into a pre-B-cell line.

We describe an immunoglobulin gene recombination indicator in which a specific rearrangement via deletion results in the acquisition of a dominant phenotype. The indicator consists of the Escherichia coli xanthine/guanine phosphoribosyltransferase (gpt) gene, whose translation is prevented by the presence of an upstream initiation codon out of frame with respect to the gpt coding sequence. Flanking this barrier initiation codon are the heptamer-spacer-nonamer recognition sequences from a kappa chain variable region (V kappa) and from a kappa chain joining region (J kappa). A proper V-J joint results in the deletion of the translational barrier and allows expression of the selectable marker. When tested by transfection into fibroblasts, no rearrangements were detected and the presence of the barrier initiation codon was sufficient to completely abolish gpt expression in these cells. Similarly, no rearrangements were detected after transfer of the test gene into myeloma cells. However, when the construct was introduced into the pre-B-cell line 38B9, greater than 80% of the transfected cells showed evidence of a specific rearrangement. These rearrangements were associated with the translation of gpt, although no selection for its expression was needed. DNA sequence analysis of six different V-J joints revealed that the rearrangement proceeded with a high degree of accuracy. These results indicate that only very minimal DNA sequences (21 base pairs 5' of the V heptamer and 4 base pairs 3' of its nonamer; less than 45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that kappa gene segments can rearrange by a deletional mechanism.

Antibody Diversity↗

PubMed2001

New isoforms of VEGF are translated from alternative initiation CUG codons located in its 5'UTR.

Vascular endothelial growth factor (VEGF) has a central role in normal as well as in tumor angiogenesis. As such, VEGF is subjected to multi-level regulation at the transcriptional, post-transcriptional, translational, and post-translational levels to ensure proper expression during embryogenesis and adulthood. Its mRNA contains an exceptionally long (1038 bp) 5' untranslated region (5'UTR), which has a role in transcriptional as well as translational regulation of VEGF expression. In this communication, we provide new evidence showing that an open reading frame (ORF) present in the 5'UTR encodes for new putative isoforms of VEGF due to alternative translational initiation from CUG codons. Like VEGF, the translation of the new isoforms is not sensitive to stress signals such as anoxia. Most likely, these isoforms either possess new capabilities, which are different from the activity of the classical VEGF isoforms, or affect the efficiency and capacity of translational initiation from the canonical AUG codon.

5' Untranslated Regions↗

PubMed2005

In vitro selection of phage RB69 RegA RNA binding sites yields UAA triplets.

The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG. Removing the 3' AUG slightly reduced binding in gel shift assays, moving the AUG 5' proximal of the (UAA) slightly improved binding, but (UAA)4 alone still bound the purified protein. Dissociation constants showed that RNA shortened to (UAA)3 and (UAA)2 also retained binding, whereas cytosine clearly prevented binding by RB69 RegA. Scanning of RB69 gene starts and ends with an RB69 RegA SELEX information weight matrix yielded 21 sequences as potential RegA sites. One site, on the mRNA for the pentameric (4:1) phage gp44/62 DNA polymerase clamp loader complex, has the RB69 gene 44 stop codon and 3'-adjacent gene 62 initiation codon in a sequence (GAAAUAAUAUG) that is similar to in vitro selected RNA and was shown to bind RB69 RegA. Sequences between the Shine-Dalgarno and initiation codon, which frequently contain a UAA stop codon of a 5'-adjacent gene, appear to be preferred RB69 RegA binding sites.

Binding Sites↗

PubMed2000

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.

Base Sequence↗

PubMed1996

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.

Bacteriophage T7↗

PubMed1987

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli.

Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5' to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5' to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.

Base Sequence↗

PubMed2003

Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting.

A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully purified individual components. This observation allowed us to demonstrate for the first time a delay in the conversion of the 48S complex into the 80S complex for beta-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit.

Animals↗

PubMed2000

Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme.

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

Animals↗

PubMed1987

Antisense oligodeoxynucleotides provide insight into mechanism of translation initiation of two Sendai virus mRNAs.

Translation of Sendai virus nucleocapsid protein (NP) and phosphoprotein (P/C) mRNAs in rabbit reticulocyte lysates in the presence of antisense oligodeoxynucleotides (15-20-mers) showed that oligonucleotides having complementarity within the 5' noncoding region of the mRNAs blocked translation, oligonucleotides having complementarity within 20 nucleotides upstream from the initiator codon blocked translation only partially, and oligonucleotides complementary to the coding region of mRNA had no effect on translation. The results suggest the possibility that the 80 S initiation complex may form about 20 bases upstream from the initiator codon. Alternatively, the 40 S preinitiation complex may recognize an initiator codon at least 20 nucleotides upstream from the codon, activating a helix-destabilizing process.

Animals↗

PubMed1992

Biosynthesis of reovirus-specified polypeptides: ribosome pausing during the translation of reovirus S1 mRNA.

The steady-state distribution of translating ribosomes on the reovirus serotype 1 Lang strain polycistronic s1 mRNA and the monocistronic s4 mRNA was mapped using a sensitive primer extension assay and cDNA clones of the S1 and S4 genes. The distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was not uniform. Major positions of ribosome pausing were detected in both rabbit reticulocyte and wheat germ cell-free protein synthesizing systems programmed with wild-type and site-directed mutant mRNAs. Two of the ribosome pause sites represent initiation and termination, rate-limiting steps of translation. For the polycistronic s1 mRNA, analysis of mutants in which either the sigma 1 ORF1 initiation codon or the sigma 1NS ORF2 initiation codon was eliminated by site-directed mutagenesis unequivocally established the identity of the specific ribosome pauses with specific ORF translational events. Ribosomes were far less uniformly distributed along the overlapping ORF regions of the polycistronic s1 mRNA than they were along the ORF of the monocistronic s4 mRNA. Furthermore, the rate-limiting initiation event at the sigma 1NS ORF2 AUG led to ribosome stacking and elongation arrest in the sigma 1 ORF1. These results begin to provide a conceptual framework for the dynamics of translation of complex as compared to simple viral mRNAs and suggest that ribosome activity may vary at multiple discrete regions on an mRNA.

Amino Acid Sequence↗

PubMed2006

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR.

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2alpha in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2alpha. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2alpha. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2alpha. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2alpha phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.

Alphavirus Infections↗

PubMed1994

Evolution of the WANCY region in amniote mitochondrial DNA.

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.

Amino Acid Sequence↗

PubMed1993

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence.

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5' leader region of brome mosaic virus (BMV) RNA4 and the goat pre-alpha-lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.

Animals↗

PubMed1998

The distance between the 3'-pyrimidine-rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA.

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5'-terminal extra-cistronic region of HAV RNA were inserted in the intergenic region of the bi-cistronic plasmid pSV-GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS-1 cells, the transient expression of both genes was quantified. The importance of the 3'-PRT appeared to be strongly influenced by the length of the 'spacer' sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the 'spacer' back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3'-PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5'-PRT fluctuated: while integrity of domain 100-106 was always stringently required for initiation to occur, the activity of domain 113-118 paralleled that of the 3'-PRT, and the opposite applied to domain 121-126, whose contribution became relevant only after switching off the 3'-PRT. Systematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.

Base Sequence↗

PubMed1998

Functional characterization of the internal ribosome entry site of eIF4G mRNA.

The eIF4 group initiation factors are required for cap-dependent translation initiation. Infection of mammalian cells by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, which severely restricts cap-dependent initiation but permits cap-independent initiation to proceed from an internal ribosome entry site (IRES) in picornaviral RNAs. The first 357 nucleotides (nt) of the 5'-untranslated region of eIF4G mRNA also contains an IRES. Using bicistronic constructs for expression in K562 cells, we have now shown that progressive deletions of the 5'-untranslated region can have either stimulatory or inhibitory effects. Furthermore, a 101-nt segment exhibits full IRES activity, and an 81-nt segment exhibits detectable IRES activity. A polypyrimidine tract (PPT) at the 3' terminus is essential for internal initiation, a property which is characteristic of picornaviral IRESs but not the other host cellular IRESs studied to date. IRES activity does not require sequences beyond 357 nt. Out-of-frame AUGs have no effect on IRES-driven luciferase expression when introduced upstream of the PPT but markedly decrease expression when introduced at sites between the PPT and the authentic initiation codon at nt 369. These results suggest that the ribosomal subunit enters at or near the PPT and then scans downstream for the initiation codon.

Codon, Initiator↗

PubMed1998

Coding sequences enhance internal initiation of translation by hepatitis A virus RNA in vitro.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Cell Extracts↗

PubMed1995

Characterization of IS1221 from Mycoplasma hyorhinis: expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift mechanism.

Seven complete and two partial copies of IS1221 variants from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae characterized to date have established a consensus IS1221 as a 1513 bp element with unique structural characteristics resembling the IS3 family of bacterial insertion sequences. Each IS1221 copy contains highly conserved 28 bp imperfect terminal inverted repeats and three distinctive internal inverted repeats (LIR, RIR and IIR). IIR is located within the coding region of the element and it is proposed that it plays a critical role in the regulation of putative transposase expression. Consensus IS1221 and one particular copy, G1135.2, contain a single long open reading frame (ORF). Two potential initiation codons are present at nucleotide 46 (AUG46) and nucleotide 397 (AUG397) and both are preceded by strong ribosome-binding sites. Both initiation codons can be used efficiently in an Escherichia coli T7 expression system. The LIR has a negative regulatory effect on translation initiation from AUG46. A-1 translational frameshift event is shown to be involved in expression of the IS1221 ORF and results in the production of 20 kDa and 6 kDa truncated proteins from the respective upstream initiation codons of the IS1221 ORF. Base substitution and deletion mutations in sequences resembling characterized motifs in documented examples of translational frameshifting resulted in a significant increase in the full-length products and a corresponding decrease in the truncated products from the IS1221 ORF. In contrast to the usual -1 frameshift regulatory event in the IS3 family, which produces a transframe fusion product as the active transposase, IS1221 may have evolved a high-frequency -1 frameshift mechanism that produces a truncated product from the upstream coding domain and thereby results in the regulated low-level production of the full-length presumptive transposase.

Amino Acid Sequence↗