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PubMed1996

Sequences directing C to U editing of the plastid psbL mRNA are located within a 22 nucleotide segment spanning the editing site.

In plastids, editing of an ACG codon to an AUG codon creates the translation initiation codon for the psbL and ndhD transcripts in tobacco. To identify the RNA segment required for psbL editing, chimeric kanamycin resistance genes were constructed containing psbL deletion derivatives, and tested in vivo for editing in transgenic plants. We report here that a 22 nucleotide segment is sufficient to direct efficient psbL editing, including 16 nucleotides upstream and five nucleotides downstream of the editing site. Mutation of the A nucleotide to a C upstream of the editing site completely abolished editing, while mutation of the downstream G to a C only reduced the editing efficiency. Out of the 22 nucleotide editing target sequence, the 16 upstream nucleotides were found to compete with the endogenous psbL transcript for a depletable trans-factor. To test whether editing of initiation codons involves a common trans-factor, a chimeric gene containing the ndhD editing site was expressed in tobacco plastids. As for psbL, editing of the ndhD site requires a depletable trans-factor. However, the ndhD trans-factor is distinct from that required for psbL editing. Distinct cissequences and trans-factor requirements for the psbL and ndhD editing sites indicate an individual recognition mechanism for the editing of plastid initiation codons.

Amino Acid Sequence↗
PubMed1980

Secondary structure of mouse and rabbit alpha- and beta-globin mRNAs: differential accessibility of alpha and beta initiator AUG codons towards nucleases.

The nucleotide sequence from the 5' terminus inward of one third of mouse alpha- and beta maj-globin messenger RNAs has been established. In addition, using 5' 32P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit alpha- and beta-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit beta-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the alpha-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with beta-globin mRNA in rabbit reticulocyte is 30--40% faster than for alpha-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.

Animals↗
PubMed2005

A single nucleotide substitution that abolishes the initiator methionine codon of the GLDC gene is prevalent among patients with glycine encephalopathy in Jerusalem.

Glycine encephalopathy (GE) (non-ketotic hyperglycinemia) is an autosomal recessive neurometabolic disease caused by defective activity of the glycine cleavage system. Clinically, patients present usually in the neonatal period with hypotonia, encephalopathy, hiccups and breath arrests with or without overt seizures. GE is considered rare, but its incidence is relatively high in several geographical areas around the world. We report a novel mutation causing GE in six extended Arab families, all from a small suburban village (population 5,000). A methionine to threonine change in the initiation codon of the glycine decarboxylase gene led to markedly reduced glycine decarboxylase mRNA levels and abolished glycine cleavage system activity.

Amino Acid Oxidoreductases↗
PubMed1990

Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man.

Based on the diversity of nucleotide sequences of cloned hepatitis B virus DNA genomes, we have predicted possible replication of genetic variants of human hepatitis B virus. This prediction is exemplified by studies of a chronic carrier of HBsAg/adw2, who lacked anti-HBc but carried exceedingly high levels of hepatitis B virus DNA in serum. Molecular characterization of a number of clones revealed a restriction map that deviated significantly from the typical pattern of the adw2 subtype, especially around the EcoRI site commonly used as a reference point. Mutations appearing consistently in the precore and core regions included (a) mutation in the precore region resulting in a termination codon after the initiation codon, (b) mutation of the core initiation codon and (c) an inframe insert of 36 nucleotides in the precore region with a new initiation site for the core protein. The 36-nucleotide insertion resulted in a new core protein with 12 extra amino acids at its amino-terminal end. A few scattered point mutations were clustered in the amino-terminal half of the core gene. Although the core protein of this hepatitis B virus variant carried immunologically detectable HBcAg, the absence of a humoral immune response to HBcAg could have been caused by previous infection with human immunodeficiency virus. This naturally occurring human hepatitis B virus variant replicated efficiently without expressing the precore region, confirming previous observations made of the artificial mutants of duck hepatitis B virus.

Adult↗
PubMed2003

Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing.

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon-anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon-anticodon interaction with AUGs located <8 nt from the 5'-end of mRNA. To understand how eIF1 plays its discriminatory role, we determined its position on the 40S ribosomal subunit using directed hydroxyl radical cleavage. The cleavage of 18S rRNA in helices 23b, 24a, and 44 by hydroxyl radicals generated from Fe(II) tethered to seven positions on the surface of eIF1 places eIF1 on the interface surface of the platform of the 40S subunit in the proximity of the ribosomal P-site. The position of eIF1 on the 40S subunit suggests that although eIF1 is unable to inspect the region of initiation codon directly, its position close to the P-site is very favorable for an indirect mechanism of eIF1's action by influencing the conformation of the platform of the 40S subunit and the positions of mRNA and initiator tRNA in initiation complexes. Unexpectedly, the position of eIF1 on the 40S subunit was strikingly similar to the position on the 30S ribosomal subunit of the sequence and structurally unrelated C-terminal domain of prokaryotic initiation factor IF3, which also participates in initiation codon selection in prokaryotes.

Animals↗
PubMed1998

Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features.

It is well known that non-coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high- and low-abundant proteins differ in the 5' untranslated regions (UTR). Statistically, 5' UTRs of low-expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5' UTRs of high-expression mRNAs. Apart from the differences in 5' UTRs, high-expression mRNAs contain stronger termination signals. Structural features of low- and high-expression mRNAs are likely to contribute to the yield of their protein products.

5' Untranslated Regions↗
PubMed1997

Characterization of a domain of the genome of BmNPV containing a functional gene for a small capsid protein and harboring deletions eliminating three open reading frames that are present in AcNPV.

The nucleotide sequence of a 1.5-kb fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been determined. An open reading frame (ORF) has been identified, which encodes a protein of a predicted molecular mass of 15 kDa (p15). Analysis of the p15 sequence revealed that it shares significant similarities with a number of previously characterized viral capsid proteins. Two mRNAs, 0.7- and 1.3-kb, are transcribed from the p15 gene. The 0.7-kb transcript appears before BmNPV DNA replication, while the 1.3-kb transcript is transcribed only after viral DNA replication. The transcription start sites for the 0.7- or 1.3-kb transcripts have been localized into two areas mapping 114 and 557/559 bp upstream of the p15 translation initiation codon, respectively, and the two transcripts appear to share a common poly(A) addition site located 114 bp downstream of the translation termination codon. Comparisons of the nucleotide sequence of the 1.5-kb BmNPV fragment with that of another isolate of BmNPV and with Autographa californica NPV (AcNPV) genomic DNA reveal that the corresponding region of AcNPV encompasses 4.1 kb of DNA and three additional ORFs that are absent from the BmNPV genome. These findings raise questions about the origin and functional relevance of the putative polypeptides encoded by the three ORFs of AcNPV.

Amino Acid Sequence↗
PubMed1998

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments.

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.

Bacillus↗
PubMed1998

Structural characterization of the virB operon on the hairy-root-inducing plasmid A4.

The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferring tumorigenic symptoms on plants upon infection by its host bacterium, Agrobacterium rhizogenes. The virB operon on pRiA4 has been sequenced and found to be composed of 11 genes, virB1 to virB11, whose products mostly appear to be associated with the cell membrane. A novel structural characteristic is frequent overlappings between the translation termination and initiation codons of adjacent genes. This is indicative of fine tuning of relative translation frequencies for each VirB protein. These results support the view that VirB multisubunit complexes provide facilities for T-DNA transfer at the bacterial cell membrane. The structural organization of the pRiA4 virB operon was essentially identical to that of the previously reported virB operons of tumor-inducing plasmids, pTiC58 and pTiA6, and the corresponding VirB proteins of the three plasmids were extremely homologous to one another. On the basis of the structural similarity of each VirB protein, the phylogenetic relationship among pRiA4, pTiC58, and pTiA6 is discussed.

Bacterial Proteins↗
PubMed2002

Transterm: a database of mRNAs and translational control elements.

Transterm is a database that facilitates studies of translation and the translational control of protein synthesis. It contains a curated collection of elements in mRNAs that control translation, and biologically relevant mRNA regions extracted from GenBank. It is organised largely on a taxonomic basis with files and summaries for each species. Global patterns that may affect translation in particular species, for example bias in the context of initiation codons (Kozak's consensus or Shine-Dalgarno sequences) or termination codons, can be detected in the consensus and information content bias summaries. Several types of access are provided via a web browser interface. Transterm defined elements may be matched in a user's sequence or in the database. Alternatively, elements can be entered by the user to search specific sections of the database (for example, coding regions or 3' flanking regions or the 3'-UTRs) or the user's sequence. Each Transterm defined element has an associated biological description with references. The database is accessible at http://uther.otago.ac.nz/Transterm.html.

Animals↗
PubMed1978

Nucleotide sequence of gene VII and of a hypothetical gene (IX) in bacteriophage M13.

A DNA fragment containing gene VII of bacteriophage M13 has been transcribed and the nucleotide sequence of this 169-nucleotides long transcript was determined by RNA sequencing methods. Additionally, the nucleotide sequence of this gene and parts of its neighbouring genes V and VIII has been determined by the dimethylsulphate-hydrazine technique. The reading frame of gene VII has been established by determining the nucleotide changes occurring in the transcripts of two amber mutants of this gene. From these combined data it is apparent that gene VII is only 99 nucleotides long and is immediately followed by the termination codon UGA. Its initiation codon AUG is separated from gene V by only a single nucleotide. It was noted that between the UGA termination codon of gene VII and the initiation codon of the next gene (gene VIII) there is space for another, hitherto unknown gene. This gene (IX) most probably codes for the small polypeptide ("C-protein") present in mature M13 phage particles.

Base Sequence↗
PubMed2003

Constitutive display of cryptic translation products by MHC class I molecules.

Major histocompatibility complex (MHC) class I molecules display tens of thousands of peptides on the cell surface, derived from virtually all endogenous proteins, for inspection by cytotoxic T cells (CTLs). We show that, in normal mouse cells, MHC I molecules present a peptide encoded in the 3' "untranslated" region. Despite its rarity, the peptide elicits CTL responses and induces self-tolerance, establishing that immune surveillance extends well beyond conventional polypeptides. Furthermore, translation of this cryptic peptide occurs by a previously unknown mechanism that decodes the CUG initiation codon as leucine rather than the canonical methionine.

3' Untranslated Regions↗
PubMed1997

Extensive RNA editing and possible double-stranded structures determining editing sites in the atpB transcripts of hornwort chloroplasts.

Three nonsense codons and an unusual initiation codon were located within the putative coding region of the atpB gene of chloroplast DNA of the hornwort Anthoceros formosae. Nucleotide sequencing of cDNA prepared from transcripts revealed extensive RNA editing. The unusual initiation codon ACG was changed to AUG and three nonsense codons were converted into sense codons. In total 15 C residues of the genomic DNA were replaced by U residues in the mRNA sequences, while 14 U residues were replaced by C residues. This is the highest number of editing events for a chloroplast mRNA reported so far. Partial editing was also shown in a cDNA clone where 23 sites were edited but six sites remained unedited, representing the existence of premature mRNA. The expected two-dimensional structure of the mRNA shows the existence of a sequence complementary to every editing site, which can produce continuous base pairing longer than 5 bp, suggesting that mispairing in the double strand is the site determinant for RNA editing in Anthoceros chloroplasts. Comparison of the cDNA sequence with other chloroplast genes suggests that the mechanism arose in the first land plants and has been reduced during evolution.

Amino Acid Sequence↗
PubMed1999

Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.

In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.

Animals↗
PubMed1990

Initiation of translation at CUG, GUG, and ACG codons in mammalian cells.

Site-directed mutants of the ACG start codon of the C' protein encoded in the polycistronic Sendai virus P/C mRNA revealed that CUG, GUG, and ACG codons initiated translation rather efficiently (10-30% of the AUG initiation) in COS-1 host cells. In addition, AUA and AUU codons initiated translation at about 5% efficiency, while UUG did not initiate translation. The sequence context of these start codons (purine residues at -3 and +4) was crucial in their recognition by the ribosome. The location of the non-AUG codons in the P/C mRNA did not play a role in its recognition by ribosomes. By using CUG, the most efficient non-AUG start codon, instead of the original ACG codon and inserting an additional upstream CUG codon in the P/C mRNA, the amount of the C' protein was increased and a novel protein was synthesized. Syntheses of an increased level of C' and the novel protein did not affect downstream initiations of the P and C proteins, suggesting that more ribosomes bind the mRNA than are actually utilized for initiation of translation.

Animals↗
PubMed1999

Alternative translation initiation of Theiler's murine encephalomyelitis virus.

DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) produce a chronic demyelinating disease in which the virus persists but has a restricted expression. We previously reported that TO subgroup strains, in addition to synthesizing the picornaviral polyprotein, use an alternative initiation codon just downstream from the polyprotein's AUG to translate an 18-kDa protein called L* that is out of frame with the polyprotein (H. H. Chen et al., Nat. Med. 1:927-931, 1995; W. P. Kong and R. P. Roos, J. Virol. 65:3395-3399, 1991). L* is critically important for virus persistence and the induction of the demyelinating disease (Chen et al., 1995; G. D. Ghadge et al. J. Virol. 72:8605-8612, 1998). We have proposed that variations in the amount of translation initiation from the L* AUG versus the polyprotein AUG may occur in different cell types and therefore affect the degree of expression of viral capsid proteins. We now demonstrate that ribosomal translation initiation at the polyprotein's initiation codon affects initiation at the L* AUG, suggesting that ribosomes land at the polyprotein's initiation codon before scanning downstream and initiating at the L* AUG. We also find that the viral 5' untranslated region affects utilization of the L* AUG. Surprisingly, mutant DA cDNAs were found to be infectious despite the presence of mutations of the polyprotein initiation codon or placement of a stop codon upstream of the L* AUG in the polyprotein's reading frame. Sequencing studies showed that these viruses had a second site mutation, converting the reading frame of L* into the polyprotein's reading frame; the results suggest that translation of the polyprotein during infection of these mutant viruses can be initiated at the L* AUG. These data are important in our understanding of translation initiation of TMEV and other RNAs that contain an internal ribosome entry site.

Animals↗
PubMed2000

[Molecular analysis of protein 4.1 gene in teratozoospermic and azoospermic patients].

In normal sperm, the 135 kD isoform of protein 4.1 is replaced by the 80 kD variant. Transcript for protein 4.1 loses the 'upstream' initiation codon by a stage-dependent alternative splicing and in mature sperm only 'downstream' initiation codon is active. A mutation in the 'downstream' initiation codon may be a reason for sperm differentiation arrest (azoospermia) or can be associated with the presence of amorphous spermatozoa in ejaculate (teratozoospermia). The aim of the study was the molecular analysis of gene coding for the protein 4.1, carrying a 'downstream' translation initiation codon. We have screened DNA samples obtained from azoospermic (blood) and teratozoospermic (spermatozoa) patients using PCR amplification of gene fragment, AUG containing exon with subsequent digestion (NlaIII) of CATG sequence. The absence of a cleavage site for this restriction enzyme would suggest the presence of mutation in the AUG codon. Analysis of DNA samples obtained from both azoospermic and teratozoospermic patients did not reveal any changes in the 'downstream' translation initiation codon. We concluded therefore that observed by others a defective expression of protein 4.1 in amorphous sperm cells is probably due to the other factor(s) than mutation in the 'downstream' translation initiation codon.

Codon↗
PubMed1984

The nifH and nifDK promoter regions from Rhizobium japonicum share structural homologies with each other and with nitrogen-regulated promoters from other organisms.

In several species of Rhizobium the three genes which encode the nitrogenase complex are separated into two operons, nifH and nifDK. We have mapped the transcriptional promoter sites for these two operons from R. japonicum USDA strain 110 by S1 protection analyses using bacterial RNA isolated from soybean nodules. Transcription of the nifDK operon is initiated at a site located 46 nucleotides upstream of the proposed translation initiation codon. nifH transcription initiates predominantly at a site 152 nucleotides upstream of the proposed translation initiation codon. An additional minor start for nifH is found 35 nucleotides downstream of the major initiation site. The nucleotide sequences of these promoter sites are presented. Comparison of the major R. japonicum nif promoter sequences reveals a high degree of sequence homology with the conserved sequences clustered in the -11 to -15, -21 to -25, -27 to -31, and -38 to -41 regions. Conservation is also observed for the -11 to -15 and -21 to -25 regions with the R. meliloti nifHDK, a cowpea Rhizobium nifH, and even Klebsiella pneumoniae nif promoters. The -27 to -31 and -38 to -42 region homologies are not conserved with the other known nif promoters. These results are discussed in light of models for nif promoter activation.

Base Sequence↗