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At least 1,639 records · Page 91Linked to original sources
PubMed1978

DNA sequence from the histidine operon control region: seven histidine codons in a row.

The DNA sequence of 250 base pairs preceding the first structural gene of the histidine operon of Salmonella typhimurium was determined by the dideoxy chain-termination method. Single-stranded DNA template was provided by an M13-histidine transducing phage constructed for the purpose by in vitro recombination. The termination site for the histidine leader RNA is identified by analogy with the trp operon leader termination sequence, and is 47 nucleotides before the start codon of the first structural gene G. Beginning 150 nucleotides before the end of the presumed leader RNA is a possible short protein-coding region with seven histidine codons in a row. It is proposed that the major mechanism of histodine operon control must involve a ribosome arrested at this run of histidine codons when histidine is limiting.

Base Sequence↗
PubMed1980

Initiation of protein synthesis in bacteria at a translational start codon of mamalian cDNA: effects of the preceding nucleotide sequence.

Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.

Animals↗
PubMed1980

Different pattern of codon recognition by mammalian mitochondrial tRNAs.

Analysis of an almost complete mammalian mitochondrial DNA sequence has identified 23 possible tRNA genes and we speculate here that these are sufficient to translate all the codons of the mitochondrial genetic code. This number is much smaller than the minimum of 31 required by the wobble hypothesis. For each of the eight genetic code boxes with four codons for one amino acid we find a single specific tRNA gene with T in the first (wobble) position of the anticodon. We suggest that these tRNAs with U in the wobble position can recognize all four codons in these genetic code boxes either by a "two out of three" base interaction or by U.N wobble.

Animals↗
PubMed1985

Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon.

Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon. Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques. The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH. This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene. The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA. This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena.

Animals↗
PubMed1985

Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon.

Proton NMR analyses have been made to elucidate the conformational characteristics of modified nucleotides as found in the first position of the anticodon of tRNA [derivatives of 5-methyl-2-thiouridine 5'-monophosphate (pxm5s2U) and derivatives of 5-hydroxyuridine 5'-monophosphate (pxo5U)]. In pxm5s2U, the C3'-endo form is extraordinarily more stable than the C2'-endo form for the ribose ring, because of the combined effects of the 2-thiocarbonyl group and the 5-substituent. By contrast, in pxo5U, the C2'-endo form is much more stable than the C3'-endo form, because of the interaction between the 5-substituent and the 5'-phosphate group. The enthalpy differences between the C2'-endo form and the C3'-endo form have been obtained as 1.1, -0.7, and 0.1 kcal/mol (1 cal = 4.184 J) for pxm5s2U, pxo5U, and unmodified uridine 5'-monophosphate, respectively. These findings lead to the conclusion that xm5s2U in the first position of the anticodon exclusively takes the C3'-endo form to recognize adenosine (but not uridine) as the third letter of the codon, whereas xo5U takes the C2'-endo form as well as the C3'-endo form to recognize adenosine, guanosine, and uridine as the third letter of the codon on ribosome. Accordingly, the biological significance of such modifications of uridine to xm5s2U/xo5U is in the regulation of the conformational rigidity/flexibility in the first position of the anticodon so as to guarantee the correct and efficient translation of codons in protein biosynthesis.

Anticodon↗
PubMed1987

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Bacteriophage lambda↗
PubMed1987

A segment of GCN4 mRNA containing the upstream AUG codons confers translational control upon a heterologous yeast transcript.

GCN4 encodes a transcriptional activator in Saccharomyces cerevisiae that is regulated at the translational level. We show that an approximately 240-nucleotide segment from the GCN4 mRNA leader containing four AUG codons is sufficient to confer translational control typical of GCN4 upon a GAL1-lacZ fusion transcript. Regulation of the hybrid transcript is dependent upon multiple positive (GCN) and negative (GCD) trans-acting factors shown to regulate GCN4 expression post-transcriptionally. This result limits the target sequences for these factors to a small internal segment of the GCN4 mRNA leader. The elimination of AUG codons within this segment substantially reduces the usual derepressing effect of mutations in five GCD genes upon GCN4-lacZ expression. This supports the idea that the products of these negative regulatory genes act by modulating the effects of the upstream AUG codons on translation of GCN4 mRNA.

Base Sequence↗
PubMed1989

Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons.

Human basic fibroblast growth factor (bFGF) is an angiogenic polypeptide mitogen present in a wide variety of mesoderm- and neuroectoderm-derived tissues. bFGF cDNA and genomic clones predict a 17.8-kDa (155-amino acid) gene product based on the presence of a single putative translational initiator ATG codon. However, a bFGF protein isolated from human placenta contains two additional amino acids NH2-terminal to the predicted initiator methionine. We report here that the human cell line SK-HEP-1 coexpresses four molecular forms (17.8, 22.5, 23.1, and 24.2 kDa) of bFGF. The 17.8-kDa bFGF protein is translationally initiated at the previously predicted methionine (AUG) codon, whereas the 22.5-, 23.1-, and 24.2-kDa proteins initiate at unusual non-AUG codons. The higher molecular weight forms are colinear NH2-terminal extensions of the 18-kDa bFGF.

Animals↗
PubMed1989

Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

Adenosine↗
PubMed1989

High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons.

A 6.75-kilobase human hepatoma-derived basic fibroblast growth factor (bFGF) cDNA was cloned and sequenced. An amino-terminal sequence generated from a purified hepatoma bFGF was found to correspond to the nucleotide sequence and to begin 8 amino acids upstream from the putative methionine start codon thought to initiate a 154-amino acid bFGF translation product. This sequence suggests that a form of bFGF of at least 163 amino acids exists. The hepatoma cDNA was transcribed in vitro into RNA; in vitro translation of this RNA generated three forms of bFGF with molecular masses of 18, 21, and 22.5 kDa. By use of in vitro mutagenesis, it was found that the 22.5-kDa bFGF and possibly the 21-kDa form were initiated with CUG start codons. The 18-kDa bFGF was initiated with an AUG codon. By transfecting into COS cells human hepatoma bFGF cDNA and a construct from which the AUG initiator was eliminated, it was found that the higher molecular mass forms of bFGF were as biologically active as the 18-kDa form.

Amino Acid Sequence↗
PubMed1991

Stop codon in the procollagen II gene (COL2A1) in a family with the Stickler syndrome (arthro-ophthalmopathy).

Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (COL2A1) was carried out in a family with the Stickler syndrome, or arthro-ophthalmopathy, an autosomal dominant disorder that affects the eyes, ears, joints, and skeleton. The analysis demonstrated linkage of the disease and COL2A1 with a logarithm-of-odds score of 1.51 at zero recombination. A newly developed procedure for preparing cosmid clones was employed to isolate the allele for type II procollagen that was linked to the disease. Analysis of over 7000 nucleotides of the gene revealed a single base mutation that altered a CG dinucleotide and converted the codon CGA for arginine at amino acid position alpha 1-732 to TGA, a stop codon. From previous work on procollagen biosynthesis, it is apparent that the truncated polypeptide synthesized from an allele with a stop codon at alpha 1-732 cannot participate in the assembly of type II procollagen, and therefore that the mutation would decrease synthesis of type II procollagen. It was not apparent, however, why the mutation produced marked changes in the eye, which contains only small amounts of type II collagen, but relatively mild effects on the many cartilaginous structures of the body that are rich in the same protein.

Base Sequence↗
PubMed1991

Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts.

We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E. coli. These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site. We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts. Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition. Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified [Bare, L.A. & Uhlenbeck, O.C. (1986) Biochemistry 25, 5825-5830] as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Translation of reovirus RNA species m1 can initiate at either of the first two in-frame initiation codons.

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.

Animals↗
PubMed1996

Amino acid residue at codon 268 determines both activity and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases. In vitro mutagenesis study.

Histo-blood group A transferase produces A antigens and transfers GalNAc to the acceptor substrate, H structures of glycolipids and glycoproteins. B transferase transfers galactose in place of GalNAc to the same acceptor substrate to synthesize B antigens. We have previously identified four amino acid substitutions between human A and B transferases. Out of these four, substitutions at the last two positions (codons 266 and 268) were found to be crucial for the different donor nucleotide-sugar specificities between A and B transferases as analyzed by gene transfer of chimeric A-B transferase genes. In the present study, we have in vitro mutagenized codon 268 of these two transferase cDNA expression constructs (glycine and alanine in A and B transferases, respectively) and produced substitution constructs with every possible amino acid residue at this position. We examined the activity and specificity of each construct by gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs on the A transferase backbone with alanine, serine, and cysteine expressed enzymes with A and B transferase activities. Weak A activity was detected with histidine and phenylalanine constructs while weak B activity was detected with asparagine and threonine constructs. All the other amino acid substitutions at codon 268 on the A transferase backbone showed neither A nor B activity. The glycine construct on the B transferase backbone expressed both A and B transferase activities. Some substitution constructs on the B transferase backbone maintained B activity while some other substitutions abolished the activity. These results show that the side chain of the amino acid residue at 268 of the human A and B transferases is responsible for determining both activity and nucleotide-sugar donor substrate specificity and strongly suggest its direct involvement in the recognition of and binding to the sugar moiety of the nucleotide-sugars.

Amino Acids↗
PubMed1996

Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome.

The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M-1 s-1 and 24 s-1 (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds form initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.

Codon↗
PubMed1999

Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA). Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon aug in oppa mRNA.

We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

5' Untranslated Regions↗
PubMed2005

The codon 47 polymorphism in p53 is functionally significant.

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent serine 46 by the proline-directed kinase p38 MAPK is known to greatly enhance the ability of p53 to induce apoptosis. Here we showed that the serine 47 polymorphic variant, which replaces the proline residue necessary for recognition by proline-directed kinases, is a markedly poorer substrate for phosphorylation on serine 46 by p38 MAPK. Consistent with this finding, we showed that the serine 47 variant has up to 5-fold decreased ability to induce apoptosis compared with wild type p53. Mechanistically, we found that this variant has decreased ability to transactivate two p53 target genes, p53AIP1 and PUMA, but not other p53 response genes; this is the first time that phosphorylation of serine 46 has been implicated in transactivation of PUMA by p53. Down-regulation of PUMA in cells with wild type p53 using short interfering RNAs reduced apoptosis in these cells to a level comparable to that in cells containing the serine 47 variant. The combined data indicated that, like the codon 72 polymorphism, the codon 47 polymorphism of p53 is functionally significant and may play a role in cancer risk, progression, and the efficacy of therapy.

Amino Acid Sequence↗
PubMed2005

Effective translation of the second cistron in two Drosophila dicistronic transcripts is determined by the absence of in-frame AUG codons in the first cistron.

The novel dicistronic transcript encoded by the Drosophila melanogaster stoned gene was recognized as being unusual in that the protein encoded by the first open reading frame, stoned-A (STNA), contains no internal methionine residues in a protein of 93 kDa. The dicistronic nature of the stoned locus and the lack of methionine residues in STNA is conserved across dipteran species. A second methionine-free cistron, encoding Snapin, was identified in Drosophila and also found to be dicistronic, the second open reading frame (ORF) encoding a methyltransferase. We have replaced the methyltransferase cistron with green fluorescent protein (GFP) and used this dicistronic construct to show that the GFP cistron is translated in Drosophila S2 cells. The insertion of in-frame AUG codons into the snapin ORF attenuates the translation of GFP, and the level of attenuation correlates with the number of inserted AUGs. Increasing the efficiency of translation-initiation of the Snapin cistron also attenuates the translation of GFP. This indicates that failure to initiate translation at the first AUG allows ribosomes to scan through the Snapin ORF and to initiate translation of the second cistron, unless new AUG codons are inserted. These data are used to interpret the expression of the stoned locus and in particular, to explain the altered stoned protein levels in the stoned-temperature-sensitive mutant allele, which replaces a lysine with a methionine codon early in the first, stonedA, cistron.

Alleles↗