In vivo analysis of the mechanisms responsible for strong transcriptional polarity in a “sense” mutant within an intercistronic region

Alifano, Pietro; Ciampi, M S; Nappo, A. G.; Bruni, Carmelo B.; Carlomagno, Maria Stella

doi:10.1016/0092-8674(88)90058-x

Cited by 30 publications

(31 citation statements)

References 58 publications

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“…This result is in keeping with the evidence provided by us for the hisC cistron (2) and that obtained for the lacZ cistron by Stanssens et al (40). It is possible that such sites might correspond to polymerase pause sites such as those described in vitro by several authors (for a review, see reference 44).…”

Section: Discussionsupporting

confidence: 78%

“…We have chosen polar mutations in the his operon of S. typhimurium as a model system to study the Rho-dependent transcription termination process (2,6). Polarity depends on the existence of a defined region(s) on the nascent transcripts that could represent the genetic signal(s) for Rho action and which is located between the nonsense codon and the next reinitiation of translation site (1,34).…”

Section: Discussionmentioning

confidence: 99%

“…The region has been identified by isolating nonpolar revertants, which turned out to have small deletions in a limited region, confirming that defined sequences are required for or are involved in the process of transcription termination leading to polarity (6). We have recently shown that it is possible to perform transcriptional analysis of the events of transcription termination in polar mutants in vivo (2).…”

mentioning

confidence: 99%

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Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium

et al. 1989

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Previous genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium

et al. 1989

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“…1). Results from several laboratories (2,22,27,38,39) support the proposal of Stanssens et al (33) that transcriptional polarity does not require complete arrest of translation, as is the case with the polarity caused by nonsense mutations (26). Increased spacing between the RNA polymerase and the leading ribosome would seem to satisfy the requirements for intracistronic termination to occur.…”

Section: Vol 177 1995 Notes 1897supporting

confidence: 71%

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box

1995

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DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box. By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes. The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene. In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase. Firstly, we analyzed the degree of derepression of the dnaA gene, measured as ␤-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids. Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box. Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression.

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“…Unlike rho-independent terminators, which usually occur between genes, rho-dependent terminators have been identified both between genes, as in AtR, (31) and trpt' (18), and within an operon, as in the his operon of Salmonella typhimurium (4,6) and the ilvGMEDA operon (32) and lacZ gene (22) of E. coli. In the latter two cases, the polarity of certain mutations has been attributed to rho-dependent termination under conditions in which translation has stopped.…”

Section: Discussionmentioning

confidence: 99%

The nature of the traK4 mutation in the F sex factor of Escherichia coli

1994

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The sequence of traK gene of the F sex factor of Escherichia coli is presented; the traK gene product is predicted to be a protein of 25,627 Da with a signal sequence of 21 amino acids to give a mature protein of 23,307 Da. The traK4 mutation is an extremely polar mutation in the F plasmid that affects F pilus synthesis and plasmid transfer. traK genes carrying the traK4 mutation and a nonpolar mutation traK105 were cloned, sequenced, and identified as an amber nonsense and a frameshift mutation, respectively. The traK4 mutation occurred within one predicted rho-dependent transcription termination element (TTE) and immediately upstream of another, while the traK105 mutation occurred after the two potential TTEs within the traK gene. S1 nuclease protection analysis and Northern (RNA) blot analysis were used to confirm that the traK4 mutation, but not the traK105 mutation, caused premature termination of transcription. Computer analysis of the F transfer region suggested the presence of TTE motifs at regular intervals throughout the 33.4-kb sequence.

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In vivo analysis of the mechanisms responsible for strong transcriptional polarity in a “sense” mutant within an intercistronic region

Cited by 30 publications

References 58 publications

Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium

Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box

The nature of the traK4 mutation in the F sex factor of Escherichia coli

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