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Results for “Codon, Terminator”

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PubMed1974

The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites.

With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.

Bacterial Proteins↗

PubMed2002

Complete ORF1b-gene sequence indicates yellow head virus is an invertebrate nidovirus.

We report the sequence of an 8503 nucleotide (nt) region of the genome of yellow head virus (YHV) encompassing the open reading frame (ORF) 1b gene. Comparison with the sequence of Australian gill-associated virus (GAV) indicated that the region, comprising approximately 30% of the YHV genome, commences 268 nt upstream of the putative ORF1a termination codon and continues through ORF1b to a site 30 nt downstream of the ORF2 initiation codon. YHV ORF1a and ORF1b overlap by 37 nt. MFOLD analysis of the overlap and downstream region predicted a 131 nt folding structure (deltaG = -47.3 kcal mol(-1)) with potential to form an RNA pseudoknot. The structure resides 3 nt downstream of a ribosomal frame-shift 'slippery' sequence (AAAUUUU) and a -1 frame-shift at this site would extend the ORF1 polyprotein by 2616 amino acids (299322 Da). In ORF1b, YHV shares 88.9% amino acid sequence identity with GAV and includes conserved polymerase, metal ion binding, helicase and other domains (Motifs 1 and 3) characteristic of nidoviruses. Compared to GAV, the YHV non-coding region linking the ORF 1b and ORF2 genes contains a 263 nt insertion. However, the region contains a conserved core sequence of 46 nucleotides (84.8% identity) that includes a stretch of 20 identical nucleotides surrounding a sub-genomic RNA transcription termination site. The data confirms the taxonomic placement of YHV in the Nidovirales and supports biological and topographical evidence that YHV and GAV may be classified as distinct species.

Amino Acid Sequence↗

PubMed1995

A mutant allele of the SUP45 (SAL4) gene of Saccharomyces cerevisiae shows temperature-dependent allosuppressor and omnipotent suppressor phenotypes.

Using a plasmid-based termination-read-through assay, the sal4-2 conditional-lethal (temperature-sensitive) allele of the SUP45 (SAL4) gene was shown to enhance the efficiency of the weak ochre suppressor tRNA SUQ5 some 10-fold at 30 degrees C. Additionally, this allele increased the suppressor efficiency of SRM2-2, a weak tRNA(Gln) ochre suppressor, indicating that the allosuppressor phenotype is not SUQ5-specific. A sup+ sal4-2 strain also showed a temperature-dependent omnipotent suppressor phenotype, enhancing readthrough of all three termination codons. Combining the sal4-2 allele with an efficient tRNA nonsense suppressor (SUP4) increased the temperature-sensitivity of that strain, indicating that enhanced nonsense suppressor levels contribute to the conditional-lethality conferred by the sal4-2 allele. However, UGA suppression levels in a sup+ sal4-2 strain following a shift to the non-permissive temperature reached a maximum significantly below that exhibited by a non-temperature sensitive SUP4 suppressor strain. Enhanced nonsense suppression may not therefore be the primary cause of the conditional-lethality of this allele. These data indicate a role for Sup45p in translation termination, and possibly in an additional, as yet unidentified, cellular process.

Alleles↗

PubMed2000

Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy.

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.

Amino Acid Sequence↗

PubMed1995

A novel mutation causing an aberrant splicing in the protein 4.2 gene associated with hereditary spherocytosis (protein 4.2Notame).

We investigated a Japanese patient with protein 4.2 deficiency. SDS-PAGE showed a complete deficiency of protein 4.2, while Western blot analysis revealed a marked decrease in the amount of protein 4.2, and the existence of a doublet of 74 and 72 kDa bands. Direct sequencing and dot-blot hybridization with allele-specific oligonucleotide probes indicated that the proband was compound heterozygous for a missense mutation in codon 142 with Ala-->Thr (GCT-->ACT) and a single nucleotide substitution (G-->A) of the first base of intron 6 (G-->A) of the protein 4.2 gene. The former is the commonest mutation observed in cases of protein 4.2 deficiency, whereas the latter is a novel mutation, located within the consensus sequence of the 5' splicing site (AGGU) (Protein 4.2Notame). RT-PCR analysis using total RNA isolated from reticulocytes of the proband revealed that the intron 6 donor site mutation causes an abnormal splicing; exon 6 is spliced out with intron 6. The abnormal mRNA has a premature termination codon, as the result of a frameshift, and this instability may lead to degradation. Thus, there is a close relation between this mutation and the molecular pathogenesis of protein 4.2 deficiency.

Base Sequence↗

PubMed1996

Phylogenetic position of the Japanese river otter Lutra nippon inferred from the nucleotide sequence of 224 bp of the mitochondrial cytochrome b gene.

A 224 bp fragment of the mitochondrial cytochrome b gene has been amplified from a 30-year-old mummy-like specimen of the Japanese river otter Lutra nippon by polymerase chain reaction (PCR). The amplified products were subcloned in the Smal site of pUC 18 and sequenced. The sequence was different from those of the congeneric Eurasian otters Lutra lutra (Latvia) and Lutra lutra (China) in 7-9 nucleotides, all of which were located at the third position of a codon and identified as transitional differences A<-->G or C<-->T. The phylogenetic analysis using the 224 bp sequences of Lutra nippon, Lutra lutra (Lativa), Lutra lutra (China), Aonyx cinerea (Asian small-clawed otter), Mustela sibirica and Mustela itatsi (weasels) supports the recent morphological study that the Japanese river otter is not a subspecies of Lutra lutra, but a distinct species, Lutra nippon. We found that Lutra nippon and Lutra lutra contain the cytochrome b-like sequences, that appear to be a pseudo-form of cytochrome b gene. The sequences are characterized by the presence of deletion and termination codons by the presence of several types of sequences with minor variations, and by the faster evolutionary rate compared with that of the mitochondrial cytochrome b gene. The genes would present in the nuclear DNA rather than in the mitochondrial DNA, as in the case of the nonfunctional cytochrome b-like sequences previously reported in a rodent.

Animals↗

PubMed1987

Direct expression of a synthetic somatomedin C gene in Escherichia coli by use of a two-cistron system.

Direct expression of a growth-promoting peptide hormone, somatomedin C/insulin-like growth factor I, which is quite difficult due to the instability of somatomedin C itself in Escherichia coli, has been achieved by the use of a two-cistron system. Assuming that basic somatomedin C might be stabilized by forming a complex with an acidic polypeptide, we constructed synthetic genes consisting of two cistrons; an acidic 93-amino-acid polypeptide was coded in the first cistron followed by a synthetic somatomedin C gene in the second cistron. The chain termination codon for the first polypeptide overlapped the initiation codon for the second polypeptide in the intercistronic region, as occurs in the polycistronic E. coli tryptophan operon, whose products are associated in multi-subunit enzyme complexes. In the expression of the resulting genetic system, recombinant somatomedin C associated with the acidic polypeptide was accumulated to high levels in the cells. After treatment of the product with acetic acid to dissociate the two components, the recombinant somatomedin C was isolated in a yield of 4.0 mg from a liter of culture broth at A600 = 1.6. It was determined to be Met-somatomedin C by chymotryptic mapping as well as amino-terminal analysis.

Amino Acid Sequence↗

PubMed1990

Overproduction of release factor reduces spontaneous frameshifting and frameshift suppression by mutant elongation factor Tu.

Mutant forms of elongation factor Tu encoded by tufA8 and tufB103 in Salmonella typhimurium cause suppression of some but not all frameshift mutations. All of the suppressed mutations in S. typhimurium have frameshift windows ending in the termination codon UGA. Because both tufA8 and tufB103 are moderately efficient UGA suppressors, we asked whether the efficiency of frameshifting is influenced by the level of misreading at UGA. We introduced plasmids synthesizing either one of the release factors into strains in which the tuf mutations suppress a test frameshift mutation. We found that overproduction of release factor 2 (which catalyzes release at UGA and UAA) reduced frameshifting promoted by the tuf mutations at all sites tested. However, at one of these sites, trpE91, overproduction of release factor 1 also reduced suppression. The spontaneous level of frameshift "leakiness" at three sites in trpE, each terminating in UGA, was reduced in strains carrying the release factor 2 plasmid. We conclude that both spontaneous and suppressor-enhanced reading-frame shifts are influenced by the activity of peptide chain release factors. However, the data suggest that the effect of release factor on frameshifting does not necessarily depend on the presence of the normal triplet termination signal.

Bacterial Proteins↗

PubMed1983

Measurement of suppressor transfer RNA activity.

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.

Animals↗

PubMed1995

Glucocorticoid receptor structure and function in an adrenocorticotropin-secreting small cell lung cancer.

ACTH secretion by tumors of nonpituitary origin is characteristically resistant to negative feedback regulation by glucocorticoids. One possible mechanism for the phenomenon could be a structural defect in the intracellular glucocorticoid receptor (GR). We studied the GR in DMS-79 cells derived from a human ACTH-secreting small cell lung cancer. Compared with control cells, DMS-79 cells were found to have greatly diminished GR ligand-binding activity and immunoreactive 94-kilodalton (kDa) GR content. Northern blot analysis revealed expression of GR transcripts that appeared to be slightly larger than those in control cells. A DMS-79 cell GR cDNA was cloned by reverse transcription/polymerase chain reaction amplification of mRNA using primers specific for full-length normal GR. The derived sequence of this full-length GR differed from the reported sequence by a single altered codon (G to A; Asn to Ser at codon 363) outside the steroid-binding domain. This N363S DMS-79 GR functioned normally to activate a target gene [mouse mammary tumor virus-chloramphenicol acetyl transferase (MMTV-CAT)] in transient transfection experiments in COS cells. Evidence for expression of a second type of GR mRNA was obtained by screening a DMS-79 cell cDNA library. This GR cDNA contained normal GR sequence up to nucleotide 2155, corresponding exactly to the end of exon 7 in the normal GR gene. The sequence appended to the GR sequences was not matched by any known sequence in DNA databases and included an in-frame termination codon after only 6 bases. The predicted truncated GR protein product (GR delta) has a mol wt of 73,740 and lacks most of the ligand-binding domain. Transient transfection of the GR delta form into COS cells did not reveal any dominant negative effect on the function of a cotransfected normal GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1988

A deletion mutation in the ApoC-II gene (ApoC-II Nijmegen) of a patient with a deficiency of apolipoprotein C-II.

The apolipoprotein C-II gene from a patient with a deficiency of apoC-II was cloned and sequenced. A single base deletion of a guanosine at position 2943 in exon three of the gene of the proband was identified by sequence analysis. This point mutation results in a shift of the reading frame and introduces a premature termination codon (TGA) at a position in the gene immediately following amino acid 17 of the mature C-II apolipoprotein. This single base deletion results in the loss of a normally occurring HphI restriction enzyme site in the apoC-II gene. Amplification of the mutant DNA sequence by the polymerase chain reaction and restriction enzyme digestion with HphI established that the patient is a homozygote for the base deletion. No apoC-II was detectable in the patient's plasma by two-dimensional gel electrophoresis and immunoblotting. We propose that the guanosine deletion is the primary genetic defect in this kindred leading to premature termination and formation of a nonfunctional truncated 17-amino acid C-II apolipoprotein which ultimately results in apoC-II deficiency.

Apolipoprotein C-II↗

PubMed1994

Structural analysis of transcripts for the protein L-isoaspartyl methyltransferase reveals multiple transcription initiation sites and a distinct pattern of expression in mouse testis: identification of a 5'-flanking sequence with promoter activity.

The gene encoding the protein L-isoaspartyl-(D-aspartyl) methyltransferase (protein carboxyl methyltransferase, PCMT) is widely expressed in bacteria and eucaryotic cells. An antisense probe encompassing the first exon of the murine PCMT gene [E. A. Romanik, C. L. Ladino, S. C. D'Ardenne, and C. M. O'Connor (1992) Gene 118, 217-222] was used in ribonuclease protection assays to identify the initiation sites for PCMT transcription in mouse testis, brain, and liver tissues. Two major initiation sites, 155-157 nucleotides (nt) and 119 nt upstream from the ATG initiation codon, were identified in all tissues in addition to several minor sites. The locations of the initiation sites in testicular RNA were confirmed using ligation-mediated 5'-rapid amplification of cDNA ends (RACE). These initiation sites are situated at the 3'-end of a 407-bp genomic sequence which is sufficient to drive the expression of a firefly luciferase gene in transient transfection assays with NIH/3T3 cells. The 407-bp sequence resembles a housekeeping gene promoter in its high G+C content, lack of a TATA box and the presence of multiple potential binding sites for the transcription factors Sp1 and ETF. Alternative splicing in the C-terminal encoding sequence and in the 3'-untranslated regions of PCMT transcripts generates three distinct classes of mRNAs which were cloned from testicular poly(A)+ RNA using 3'-RACE. Transcript splicing either 38 nt downstream or 7 nt upstream from the termination codon in exon 7 produces mRNAs encoding PCMT isozymes with -RWK or -RDEL, respectively, at their C-termini. The predominant transcript in testis, which is not detected in somatic tissues by Northern blotting and which may be specific to germ cells, is not spliced within exon 7 and also encodes the -RWK isozyme.

3T3 Cells↗

PubMed1988

Evolutionary implication of genetic code deviations.

We formulate the following hypothesis: Life's origin may have occurred during the lower Archaean at a time when the environmental temperature was higher than it is at present. Preliminary consequences of this hypothesis are studied from the point of view of molecular evolution. We restrict our attention to implications regarding the genetic code. We conclude that alternative assignment of termination codons may be understood in terms of: (a) the elevated temperatures to which the progenote may initially have been exposed; and (b) the subsequent response of its genome to the opportunity provided by the eventual loss of hyperthermal genetic expression during a thermal transition (TT) period, which was triggered off by the evolution of the dynamic Earth.

Biological Evolution↗

PubMed1992

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.

Base Sequence↗

PubMed1985

The complete sequence of the chicken alpha-cardiac actin gene: a highly conserved vertebrate gene.

We sequenced the entire chicken alpha-cardiac actin gene. A single intron was positioned 20 bp upstream from the initiation ATG codon in the 5' non-coding region while the coding region was interrupted by 5 introns at amino acid positions 41/42, 150, 204, 267, and 327/328. Sequencing allowed the first comparison of the alpha-cardiac and alpha-skeletal actin transcriptional promoters. These highly G+C rich promoters share two regions of homology which are found at position -134 (10 bp) and -296 (12 bp) in the alpha-cardiac actin promoter. A smaller 9 bp motif (CCGCGCCGG) homologous to the -134 sequence was detected before, between and after the TATA and CAAT boxes of the alpha-cardiac actin gene. The polyadenylation signal (AATAAA) was located 156 bp downstream from the translation termination codon. The complete length of the alpha-cardiac actin mRNA excluding the poly A tail is 1370 nucleotides. The 3' noncoding transcribed portion of the chicken alpha-cardiac actin gene was found to be extraordinarily conserved when compared to the human and rat alpha-cardiac actin mRNA sequences.

Actins↗

PubMed1993

Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes.

The open reading frame of the human T-cell leukemia virus type II pro gene is arranged at a -1 position relative to the gag gene. Synthesis of the Gag-Pro fusion polyprotein is facilitated by ribosomal frameshift into the reading frame of the pro gene. Cloning of a synthetic 41-bp oligonucleotide corresponding to the gag-pro junction within a heterologous gene (nef of human immunodeficiency virus type I) and mutation analysis revealed that two cis-acting signals, an adenosine residue stretch and a dyad symmetry sequence, flanking the UAA termination codon, are required for efficient ribosomal frameshifting between gag and pro. The stability of the stem-loop structure is crucial for frameshifting.

Amino Acid Sequence↗

PubMed2003

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization.

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5'-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.

Amino Acid Sequence↗

PubMed1993

Functional analysis of mRNA 3' end formation signals in the convergent and overlapping transcription units of the S. cerevisiae genes RHO1 and MRP2.

The Saccharomyces cerevisiae genes RHO1 and MRP2 are convergently transcribed, with 281 base pairs separating their termination codons. Transcript mapping revealed at least 111 base pairs within the RHO1-MRP2 intercoding region are transcribed in both directions. Transplacement experiments showed distinct sequences of 70 nt for MRP2 and 179 nt for RHO1 were sufficient for normal mRNA 3' end formation. The MRP2 signal functioned in either orientation, although relatively inefficiently in the non-native orientation. This element contains a polyAT sequence essential for 3' end formation in both orientations. RHO1 or MRP2 3' end formation was not affected by overproduction or elimination of the complementary, natural antisense transcript. In contrast, insertion of a strong promoter that extended antisense transcripts beyond their normal 3' ends inactivated either MRP2 or RHO1. These data suggest that transcript termination in the compact yeast genome can be important to prevent inactivation of downstream genes as a result of antisense transcription.

Base Sequence↗