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PubMed1989

Reversion of trpA nonsense mutations by deletion of the chain-termination codons.

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.

Base Sequence↗

PubMed1987

Theoretical analysis of single-round transcription experiments on trp leader region.

A kinetic model is proposed to reproduce the time courses of the concentration change in paused leader RNA, terminated leader RNA, and readthrough RNA in the single-round transcription experiments on trp leader region of Escherichia coli and its mutants, L132, L75, and L75L135 (Winkler, M. E., and C. Yanofsky, 1981, Biochemistry, 20:3738-3744; Fisher, R., and C. Yanofsky, 1983, J. Biol. Chem., 258:9208-9212). This model fits the experimental results well and also captures the essential aspects of the processes of transcriptional pausing and termination. In the wild type template, under optimal conditions, it is found that the transcription rate at the pause and attenuation sites is of the same order of magnitude, 10,000-fold lower than the transcription rate at the other sites, and the high termination level at the attenuation site is attributable to a higher dissociation rate. This analysis also provides a clue as to how the template base change, various concentrations of ribonucleoside triphosphates, and the presence or absence of L-factor affect the transcription and dissociation rates to yield different termination levels at the pause or attenuation site. It also discusses the molecular mechanism of the transcriptional pausing and termination.

DNA-Directed RNA Polymerases↗

PubMed1994

RNA chain elongation and termination by mammalian RNA polymerase III. Analysis of tRNA gene transcription by imposing a reversible factor-mediated block to elongation using a sequence-specific DNA binding protein.

We have used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3' end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle. lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.

Base Sequence↗

PubMed1995

Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

Alkaloids↗

PubMed1990

The RNA antiterminator causes transcription pausing in the leader region of the tryptophan operon.

In vitro transcription studies with a trp leader DNA template derived from a double deletion mutant of Serratia marcescens revealed that the transcription complex pauses synthesis of part of the RNA antiterminator, structure 2:3. Pausing was enhanced by NusA protein and was dependent on the concentration of UTP in the transcription reaction mixture. A weak antiterminator pause also was detected during transcription of the wild-type S. marcescens trp leader template in the presence of NusA protein and 1 microM UTP. Transcription pausing following synthesis of the antiterminator also was observed in a cell-free transcription-translation system. Antiterminator-induced pausing may play an important role in vivo by delaying synthesis of RNA segment 4. This delay may influence basal level control in cells with an excess of tryptophan. In addition, formation of the antiterminator pause structure may introduce a more stringent tryptophan starvation requirement for RNA polymerase to read through the attenuator.

Base Sequence↗

PubMed1991

dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli.

dif (deletion induced filamentation) is a newly identified locus that lies within the terminus region of the Escherichia coli chromosome. The Dif phenotype was characterized by a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system. Interactions between dif-carrying plasmids as well as between such plasmids and the bacterial chromosome demonstrated that dif is a cis-acting, recA-independent recombination site. Filamentation continued in dif mutants in which SOS-associated division inhibitors were inoperative, which showed that induction of these inhibitors was not the primary cause of filamentation. Filamentation was not observed in dif recA or dif recBC mutants, which were unable to carry out homologous recombination. The dif site shows homology with the cer site of plasmid ColE1, which resolves plasmid multimers to monomers. It is proposed that dif functions to resolve dimeric chromosomes produced by sister chromatid exchange, and that the Dif phenotype is due to the inability of these mutants to resolve multimers prior to cell division.

Base Sequence↗

PubMed1994

Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base.

The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination. Each gene in the group is specifically induced by limitation for the appropriate amino acid. This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure. The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator. In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences. Mutations in the acceptor end of tRNA(Tyr) could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing. This base pairing may thereby directly affect the formation and/or function of the antiterminator. The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA(Tyr), was shown to act as a second specificity determinant for assuring response to the appropriate tRNA. Furthermore, overproduction of an unchargeable variant of tRNA(Tyr) resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system.

Bacillus subtilis↗

PubMed1995

The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.

Base Sequence↗

PubMed1994

Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity.

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.

Bacteriophage M13↗

PubMed1993

The 3' codon context effect on UAG suppressor tRNA is different in Escherichia coli and human cells.

We have compared the effect of 3' context on the efficiency of nonsense suppressor tRNAs in Escherichia coli and human cells. Plasmids containing amber (UAG) termination codons were constructed in the vector pRSV beta gal by oligonucleotide insertion at an N-terminal location in a lacZ fusion. A family of identical vectors was prepared with either A, C, G or U as the first 3' base following the stop codon. These derivatives of pRSV beta gal were expressed in E. coli as stable plasmids, or transiently in human 293 cell tissue culture. Nonsense suppression was monitored using enzyme assays for beta-galactosidase. In E. coli the efficiency of a plasmid-borne bacterial tRNA(trp) UAG suppressor varied A > G > C = U. When the same lacZ reporter vectors were cotransfected with a human tRNA(ser) UAG suppressor plasmid into human cells, context effects of a different nature were detected. Double reciprocal analysis of dose-response experiments were used to show that the efficiency of suppression varied C > G > U = A. The discovery of different codon context effects on nonsense suppression in human cells suggest that the interaction between mammalian tRNAs or release factors and their target codons may have different characteristics from those in bacteria.

Base Sequence↗

PubMed1999

Characterization of wild lambdoid bacteriophages: detection of a wide distribution of phage immunity groups and identification of a nus-dependent, nonlambdoid phage group.

Temperate phages were isolated from fresh human fecal samples. Lambdoid phages were screened for growth on Nus+ but not Nus- bacteria. Approximately 100 independent lysogens of Nus-dependent phages were constructed and tested for immunity to superinfection by the same Nus-dependent phages. This identified 20 different phage immunity groups, 18 of which belonged to the lambdoid phage family. The DNA from the majority of these phages hybridized with a lambda DNA probe, and approximately 50% were recognized by anti-lambda antibodies. Furthermore most were inducible by UV light. Eleven phage recombinants with different immunity were obtained when a phage from each group was coinfected with lambda or its derivative lambdaBLK20. We also identified another immunity group with 48 members. None of these hybridized with either lambda or phi80 DNA probes nor were they recognized by anti-lambda serum. Most were not induced by UV light treatment, and no recombinants were obtained when crossed with either lambda or lambdaBLK20. Consequently, this group of Nus-dependent phages represent a new nonlambdoid phage family.

Animals↗

PubMed1995

Functional analysis of mutations in the transcription terminator T1 that suppress two dnaG alleles in Escherichia coli.

The mutations dnaG2903 and parB are both temperature-sensitive conditional lethal alleles of the Escherichia coli dnaG gene, which encodes the protein primase. The lesions are located in the 3' end of the gene, 9 basepairs apart, and both cause Glu-to-Lys substitutions in the carboxy terminus of primase. Previously, it was shown that dnaG2903 can be suppressed by point mutations in the rho-independent transcription terminator T1, which is located just upstream of dnaG in the rpsU-dnaG-rpoD macromolecular synthesis operon. We report here that parB can also be suppressed by point mutations in T1, demonstrating that parB can be suppressed in the same manner as dnaG2903. We also identified additional suppressors of dnaG2903 that are point mutations in T1, suggesting that defective transcription termination leading to overexpression of dnaG2903 and parB suppresses the temperature-sensitive phenotype of strains harboring these mutations. Utilizing two mutant rpoB alleles whose transcription termination phenotypes at rho-independent terminators have been previously characterized, we demonstrate that defective transcription termination leading to the overexpression of dnaG does indeed suppress dnaG2903 and parB. The point mutations in T1 identified in this study were analyzed for their effects on termination efficiency at T1. Our results indicate that the thermodynamic stability of the hairpin structures may not be the sole determinant of termination efficiency in vivo.

Alleles↗

PubMed1989

A new procedure for the targeted inactivation of essential bacterial genes.

A new method is described for the exchange of a plasmid encoded mutant gene with its chromosomal counterpart. The method is based on positive selection and is applicable for the exchange of essential genes. The main features of the method are: (1) cloning of an antibiotic resistance marker (without its promoter) downstream of the cloned target gene, thus forming an artificial operon; (2) inactivating the target gene (and consequently also the antibiotic resistance gene) by inserting a strong transcriptional termination signal into it; and (3) selection for double recombinants by means of the antibiotic resistance gene.

Blotting, Southern↗

PubMed1989

Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants.

We have mapped three Escherichia coli RNA polymerase mutations selected by Guarente (1979) to suppress the termination defects of rho201. We find that two of the mutations are located in the 3' half of the rpoB gene encoding the beta subunit. The third mutation is in the rpoC gene, encoding the beta' subunit. All three RNA polymerase mutations affect termination efficiency, even in rho+ strains, suggesting that the C-terminal end of the beta as well as the beta' subunit participates in termination. In addition we find that all three rpoBC alleles inhibit lambda N-mediated antitermination at 30 degrees C in a strain containing the nusA1 allele. It may be significant that the three other RNA polymerase mutations known to revert the termination defect of mutant rho alleles also affect N-mediated antitermination in nusA1 strains. The correlation of these two phenotypes suggests that both phenotypes may arise from the same functional defect in RNA polymerase.

Alleles↗

PubMed1996

Suppression of the Bgl+ phenotype of a delta hns strain of Escherichia coli by a Bacillus subtilis antiterminator binding site.

Bacillus subtilis, like Escherichia coli, possesses several sets of genes involved in the utilization of beta-glucosides. In E. coli, all these genes are cryptic, including the genes forming the bgl operon, thus leading to a Bgl- phenotype. We screened for B. subtilis chromosomal DNA fragments capable of reverting the Bgl+ phenotype associated with an E. coli hns mutant to the Bgl- wild-type phenotype. One B. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from the bgl gene. Deletion studies as well as subcloning experiments allowed us to prove that the putative B. subtilis of the E. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein of E. coli bgl operon by our putative B. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.

Bacillus subtilis↗

PubMed1987

nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites.

We have studied the factors that affect transcription termination in vitro at the tR2 terminator of bacteriophage lambda and at the T1 terminator of the Escherichia coli rrnB operon. Termination efficiency at both of these sites is enhanced by the E. coli nusA protein, giving final efficiencies of termination in vitro comparable to those estimated in vivo. Transcripts terminated in the presence of nusA protein are all released from the RNA polymerase complex, indicating that a complete termination reaction is involved, rather than simply induction of a long pause at the terminator. The termination factor activity of the nusA protein does not depend on the presence of rho protein and is not detectably enhanced by that factor. Thus, the nusA protein appears to play a pleiotropic role in E. coli transcription, serving as an antitermination factor, RNA polymerase subunit and true termination factor for some terminator sites.

Bacterial Proteins↗

PubMed1987

Transcription termination sites at the distal end of the leu operon of Salmonella typhimurium.

Transcription terminates at two different sites at the distal end of the leucine operon of Salmonella typhimurium. The first of these sites (leut), located 140 base-pairs past the end of leuD, contains a G + C-rich palindrome followed by a run of T residues in the non-coding strand. Termination at leut, both in vitro and in vivo, is independent of rho protein, but is stimulated by the NusA protein. The second termination site (leut'), located 145 base-pairs beyond the first, is rho-dependent both in vitro and in vivo, and is not influenced by NusA protein. The organization of transcription termination sites at the distal end of the leu operon (a rho-independent site followed by a rho-dependent site) is similar to that for the trp operon of Escherichia coli.

Acetyltransferases↗

PubMed1991

cis Acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus.

The pol gene of the Moloney murine leukemia virus (M-MuLV) is expressed as a Gag-Pol fusion protein through an in-frame suppression of the UAG termination codon located between the two genes. The role of nucleotide context in suppression was investigated, in a rabbit reticulocyte lysate translation system, using site-directed mutagenesis. The results indicate that the translational readthrough is mediated by at least 50 bases long RNA sequence located 3' to the gag UAG termination codon. Within this sequence a short purine-rich sequence adjacent to the amber codon, highly conserved among different retroviruses, appears essential for M-MuLV suppression. Two alternative putative stem and loop like RNA structures can be drawn at the gag-pol junction, one abutting the gag UAG codon, and the second downstream to it. None of these structures appears to be important to the suppression process.

Animals↗