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At least 595 records · Page 33Linked to original sources
PubMed1994

Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity.

It has been shown that two of the three adeno-associated virus type 2 capsid proteins, B and C, are synthesized from a single spliced transcript. Protein C arises from an AUG codon at nucleotide 2810, whereas protein B is initiated by a unique eucaryotic initiation codon (ACG) that lies 65 triplets upstream from the C origin. The third capsid component, protein A, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. In this study we used oligonucleotide-directed mutagenesis to confirm the positions of the B initiation codon and the 3' acceptor sites for the alternatively spliced B/C and A protein messages. We also located definitively the protein A initiation codon, an AUG triplet mapping to nucleotide 2203. Mutagenesis of the B initiator permitted a direct test of the effect of increased B initiator strength on the translational efficiencies of the B and C proteins. It was found that conversion of the relatively inefficient protein B initiator (ACG) to an AUG enhanced the level of B synthesis while abolishing the synthesis of C from its downstream AUG initiator. Protein C synthesis thus depends on the strength of the B initiator, i.e., the relatively higher levels of C (approximately 20-fold greater than B) must result from frequent readthrough of the weak B initiator. Finally, we examined the abilities of mutants deficient in the synthesis of A, B, or C to produce infectious virions. We found that at least two of the structural proteins, B and C, are required for the production of infectious virions and that sequestration of single-stranded adeno-associated virus genomes from the pool of replicating DNA molecules does not occur in the absence of either of these proteins.

Blotting, Northern↗
PubMed1995

Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs.

The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5'-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genus Tombusvirus and Carmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.

Base Sequence↗
PubMed1989

Complete nucleotide sequence of the cucumber necrosis virus genome.

The complete nucleotide sequence of the cucumber necrosis virus (CNV) genome has been determined. The genome is 4701 nucleotides in length and contains five long open reading frames (ORF). ORF1 begins at the first AUG codon at the 5' terminus and terminates at an amber codon. The predicted molecular weight of the polyprotein encoded by ORF1 is 33 kilodaltons (kDa). Readthrough of the ORF1 amber codon would yield a protein with a molecular weight of 92 kDa. Comparison of the amino acid sequence of the 92-kDa protein with the putative replicases of carnation mottle virus (CarMV) and barley yellow dwarf virus (BYDV) shows extensive sequence similarity. This suggests that the CNV 92-kDa protein is the viral replicase and, furthermore, suggests a close evolutionary relationship between CNV, CarMV, and BYDV, members of the Tombus-, Carmo-, and Luteovirus groups, respectively. Immediately following the 92-kDa protein is ORF3 which can encode a 40-kDa protein. It is identified as the coat protein based on its similarity in amino acid composition to the previously determined CNV coat protein sequence (J. H. Tremaine, 1972, Virology 48, 582-590) and on its amino acid sequence similarity with the tomato bushy stunt virus coat protein. Two nested ORFs (ORF4 and -5), in different frames, follow the coat protein gene. Although it is not known if both ORFs are expressed, they would encode proteins with predicted molecular weights of 21 and 20 kDa, respectively.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence context effect of a cyclobutane pyrimidine dimer upon RNA polymerase II transcription.

We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA6.dT6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.

Animals↗
PubMed1989

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence and genomic organization of melon necrotic spot virus.

Cloned cDNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been sequenced and the sizes and locations of predicted viral proteins have been deduced. The genome consists of at least 4262 nucleotides and the positive strand contains three to five open reading frames (ORFs) which may be expressed. The 5' proximal ORF encodes a 29K protein (p29) and terminates with an amber codon which may be read through to produce an 89K protein (p89). Two small centrally located ORFs each encode a 7K protein (p7A and p7B). As p7A is in frame with p7B, readthrough of the amber codon terminating p7A may occur, producing a 14K protein (p14). The 3' proximal ORF encodes the 42K coat protein. The genomic organization of MNSV, its probable translation strategy and the amino acid sequences of its putative proteins closely resemble those of known carmoviruses, suggesting that MNSV should be classified as a member of the carmovirus group. Unusual properties of the putative MNSV replicase (p89) suggest that MNSV should be classified in a new virus supergroup with several other viruses sharing these properties.

Amino Acid Sequence↗
PubMed2004

Characterization of cis-acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone-inducible enterococcal plasmid pCF10.

The pCF10-encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis-acting mutations in the prgXQ region. Determination of the 3' end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3' from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone-insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.

Bacterial Proteins↗
PubMed1991

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper.

The entire genomic RNA of a Spanish isolate of pepper mild mottle virus (PMMV-S), a resistance-breaking virus in pepper, was cloned and sequenced and shown to be similar to other tobamoviruses in its genomic organization. It consisted of 6357 nucleotides (nt) and contained four open reading frames (ORFs) which encode a 126K protein and a readthrough 183K protein (nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a 17.5K coat protein (nt 5685 to 6158). This is the first tobamovirus in which none of the ORFs overlap. Both its nucleic acid and predicted protein sequences were compared with the previously determined sequences of other tobamoviruses. The variations and similarities found and their relationship with the pathogenicity of this virus are discussed.

Amino Acid Sequence↗
PubMed1989

Modified model for the switch from Sendai virus transcription to replication.

RNase mapping was used to estimate the levels of unencapsidated Sendai virus plus-strand RNAs which cross the leader-NP junction relative to NP mRNA. Significant amounts of leader readthrough RNAs were found in Z strain-infected cells, similar to that described for the polR mutant of vesicular stomatitis virus, even though this strain is considered wild type. The levels of the readthrough RNAs detected fell sharply when progressively longer probes were used, unlike that of NP mRNA. These studies suggest that polymerases which read through the first junction terminate shortly afterwards in the absence of concurrent assembly of the nascent chain, whereas those which reinitiate at NP continue efficiently to the next junction. Reinitiation appears to be necessary to convert the polymerase to a mode in which elongation is independent of concurrent assembly. Concurrent assembly appears to be required not only for the polymerase to read through the junction efficiently, but also for it to continue elongation between junctions.

Cell Line↗
PubMed1998

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association.

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3'-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3'-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Binding Sites↗
PubMed1994

The protein p30, encoded at the gag-pro junction of mouse mammary tumor virus, is a dUTPase fused with a nucleocapsid protein.

A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30. The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues. The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources. In this paper, we demonstrate that p30 is a functional dUTPase. Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough. By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed. The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity. Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits. MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase.

Amino Acid Sequence↗
PubMed1990

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus.

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus (TM-GMV) was determined. It shows 64.4% sequence homology with the genomic RNA of tobacco mosaic virus (TMV) and 66.0% with that of tomato mosaic virus (ToMV). Its genomic organization is similar to that of TMV and ToMV. The 5' proximal open reading frame (ORF) encodes a 126K polypeptide and a 183K readthrough product in which nucleotide-binding and polymerase-sequence motifs are found. The third ORF encodes a 28.5K protein homologous to TMV and ToMV movement proteins. A conserved core is found with four other tobamoviruses and two tobraviruses suggesting a common mechanism of cell-to-cell movement for tobamo- and tobraviruses. The fourth ORF encodes the 17.5K coat protein. Homology between the RNAs of TMGMV and its satellite virus STMV is limited to their 3' termini, and structural comparisons suggest that this region may determine the nature of the satellite/helper virus interaction.

Amino Acid Sequence↗
PubMed2006

An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation.

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.

Base Sequence↗
PubMed1989

Ribosomal RNA operon anti-termination. Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms.

All Escherichia coli rrn operons show a common motif in which anti-terminator box B-box A sequences occur twice, first in the leader and again in the 16 S-23 S spacer. In this study we have analyzed several aspects of rrn anti-termination by leader and spacer anti-terminator sequences. Using DNA synthesis and a plasmid test system, we incorporated random changes into the leader anti-terminator region and examined these mutations for their ability to read through a strong terminator. We also examined anti-termination by synthetic box A and by rrn spacer region sequences. Information derived from these experiments was used to search the rrn sequences of other micro-organisms for possible anti-termination features. Our principal conclusions were that: (1) box A was sufficient for terminator readthrough; (2) we could show no positive requirement for box B in our test system; (3) many of the negative anti-terminator mutations caused a promoter up-effect in the absence of a terminator; (4) the search of rrn operons from other micro-organisms revealed that anti-terminator-like box B-box A sequences exist in leader and spacer regions of both eubacteria and archaebacteria. The frequent occurrence of this pattern suggested that the E. coli rrn anti-termination motif is widespread in nature and has been conserved in microbial evolution.

Base Sequence↗
PubMed2000

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

Animals↗
PubMed1985

Negative control of oriC plasmid replication by transcription of the oriC region.

We have demonstrated that the replication of the oriC plasmid, carrying the replication origin of the Escherichia coli chromosome, is inhibited by transcriptional readthrough from an oriC flanking region of the plasmid. This was drawn from an examination of the replication of an oriC plasmid, pKZ4, which bears the lacOP segment at the right-hand side of oriC (the asnA side) in such an orientation that transcription from the lac promoter proceeds towards oriC. Replication of pKZ4 was found to be drastically inhibited by inducing transcription from the lac promoter with IPTG, an inducer of the lactose operon. When trp transcription attenuator termination sequences were inserted near the right-hand end of the oriC region of pKZ4, the replication of the plasmid became considerably insensitive to the inhibitory effect of IPTG. This indicates that the inhibition is due to the frequent leftward transcription, which initiates at the lac promoter and proceeds into the oriC region. Since IPTG inhibits the replication of pKZ4, but not that of another coexisting oriC plasmid which is devoid of the lacOP segment, the replication inhibition is judged to act only in cis. Transcription from the promoter of the chloramphenicol resistance gene also caused the inhibition of oriC plasmid replication.

Base Sequence↗
PubMed1994

Complete nucleotide sequence of peanut clump virus RNA 1 and relationships with other fungus-transmitted rod-shaped viruses.

The complete nucleotide sequence of RNA 1 of the tentative furovirus peanut clump virus (PCV) has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 5897 nucleotides in length and contains three long open reading frames (ORFs). The 5'-terminal proximal ORF has the potential to encode a polypeptide of M(r) 130942 (P131) containing methyltransferase and RNA helicase homologous domains and displaying homology with large nonstructural proteins of alpha-like viruses, which are known or thought to be involved in virus replication. The P131 ORF is followed in-frame by a second ORF which is probably expressed by partial readthrough of the UGA termination codon of the P131 ORF to produce a polypeptide of M(r) 191044 (P191). The readthrough region of P191 contains the characteristic 'core' RNA polymerase motif, indicating that the PCV replicase proteins are expressed as a pair of overlapping proteins as in the tobamoviruses, tobraviruses and the furovirus soil-borne wheat mosaic virus (SBWMV). Sequence comparisons indicate that P131 and P191 are most closely related to the replicase proteins of SBWMV and the hordeivirus barley stripe mosaic virus (BSMV) but are only distantly related to the replicase of the furovirus beet necrotic yellow vein virus (BNYVV). The 3'-terminal proximal ORF can encode a putative polypeptide of M(r) 14556 (P15) which displays homology to small cysteine-rich proteins of hordeiviruses and SBWMV. We have corrected four errors in the sequence of PCV RNA 2 published previously by Manohar et al. (Virology 195, 33-41, 1993). One of these changes causes two small ORFs near the 3' terminus of RNA 2 to be fused together to create an ORF for a putative polypeptide of M(r) 16833 (P17) which displays extensive homology with the third protein of the triple gene block of BSMV RNA beta.

Amino Acid Sequence↗
PubMed1992

Codon context effect in virus translational readthrough. A study in vitro of the determinants of TMV and Mo-MuLV amber suppression.

To assess the role of codon context on the efficiency of eukaryotic suppression of termination codons, we have compared, in a rabbit cell-free translation system, the readthrough efficiency related to two synthetic transcripts differing by the codon context around an amber codon. The codon contexts are derived from tobacco mosaic virus (TMV) and Moloney murine leukemia virus (Mo-MuLV) RNAs. The Mo-MuLV-like codon context does not promote suppression. Substituting TMV-derived triplets in the Mo-MuLV-like codon context shows that the two codons downstream from the TMV UAG signal are important determinants of suppression, as recently demonstrated in vivo.

Amino Acid Sequence↗