Effect of rifampicin on expression of<i>lacZ</i>fused to promoters or terminators of the<i>E.coli rpoBC</i>operon

Howe, Kathy; Newman, Andrew; Garner, Ian; Wallis, Anne E.; Hayward, Richard S.

doi:10.1093/nar/10.22.7425

Cited by 16 publications

(6 citation statements)

References 27 publications

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“…This observation is similar to the finding that the termination efficiency of a Rho-independent terminator also significantly varied when different nonribosomal promoters were compared by using an operon fusion assay system (13). It is not yet clear whether termination efficiency is dependent on the promoters examined or whether these observations reflect problems inherent in the use of operon fusions (see below).…”

supporting

confidence: 76%

Antitermination mechanisms in rRNA operons of Escherichia coli

Morgan¹

1986

J Bacteriol

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In this review I assess the available information about antitermination mechanisms in rRNA operons (rrn operons) of Escherichia coli. The reader is referred to reviews on rrn operon structure (18) and lambda antitermination (6) for references supporting statements on these topics.Why rrn operons have antitermination mechanisms. The idea that rrn operons might have antitermination mechanisms evolved from studies of transcriptional polarity in operons that code for translated mRNAs. Transcriptional polarity is known to be caused by premature transcription termination resulting from the uncoupling of transcription and translation when translation is interrupted by premature nonsense codons or by other mechanisms (1). The essence of coupling is that ribosomes translating an mRNA concurrent with mRNA synthesis act, in a way not yet clear, to prevent transcription termination at termination sites within proteincoding regions. Termination at sites in protein-coding regions has been found to be largely or solely Rho dependent in all cases examined. Presumably, Rho-dependent termination signals are frequent in sequences that have not specifically evolved as termination signals.The phenomenon of polarity demonstrates that coupling is required for efficient end-to-end transcription of mRNA. However, 6,000-base-pair rrn operons are transcribed endto-end without detectable transcription termination despite the fact that coupling during transcription of rrn operons is unlikely because rrn transcripts are not translated. These facts led to early experiments to demonstrate that antitermination mechanisms in rrn operons compensate for the inability of coupling to reduce premature termination (17). rrn operon structure. The promoter-leader regions of the seven E. coli rrn operons each consists of two highly conserved tandem promoters (Pi and P2) separated by 109 to 119 base pairs, followed by a highly conserved, transcribed but untranslated leader region that is 171 to 173 base pairs in length from the start of P2 transcripts to the start of the 16S rRNA gene. Between the 16S and 23S genes is a region of 350 to 450 base pairs (the spacer region) containing precursor-specific sequences and one or two tRNA genes. The 23S rRNA genes are followed by 5S rRNA genes and, in some cases, one or two tRNA genes. As discussed below, both the leader and spacer regions are suspected of being involved with antitermination.Evidence for rrn antiternmination. Early evidence for rrn antitermination was obtained when insertions of Tn9, TnlO,and IS] were observed to cause far less transcriptional polarity in rrn operons than in protein-coding operons (4,17,26). These experiments indicated that transcription termination was reduced in rrn operons. It was suggested that modifications of RNA polymerase to reduce termination could be responsible for the low polarity of these insertions, but it was also pointed out that the frequent transcription of rrn operons, structural properties of rrn transcripts, or association of rRNA with ribosomal proteins could...

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supporting

confidence: 76%

Antitermination mechanisms in rRNA operons of Escherichia coli

Morgan¹

1986

J Bacteriol

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“…380 nucleotides upstream of rpli (37,39). However, when segments of the operon were analyzed either by cloning them upstream of lacZ on a plasmid (2,5,21) or transducing phage (20) or directly by cloning onto a lambda transducing phage and examining the synthesis of the gene products in the UVirradiated cell system (28), it was suggested that there were internal promoters preceding rplL and rpoB. Most of these experiments indicated that these promoters were relatively weak.…”

Section: Resultsmentioning

confidence: 99%

Relative activities of the transcriptional regulatory sites in the rplKAJLrpoBC gene cluster of Escherichia coli

Ralling¹,

Linn²

1984

J Bacteriol

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The pattern of transcription of the rplKAJLrpoBC gene cluster of Escherichia coli appears to be complex. At least four different promoters and a transcriptional attenuator have been identified. To compare the relative effect of each of the putative promoters and the attenuator on transcription of these genes, we fused these regulatory sites to lacZ. These transcriptional fusions were constructed on lambda transducing phages so a single copy of each could be stably integrated into the chromosome. The level of ,B-galactosidase in a lysogen of each phage reflects the activity of the transcriptional regulatory site. We find that the promoters preceding rplK (rplKp) and rplJ (rplJp) are indeed the major promoters of this gene cluster. The minor promoter before rplL (rplLp) is much weaker and contributes little to the transcription of the downstream genes. Under these conditions, we find no evidence of a promoter (rpoBp) in the rplL-rpoB intercistronic region. The attenuator (atn) terminates ca. 70o of the transcripts initiated at the promoters preceding it. Although we cannot rule out that some transcripts from rplKp may read through into rplJLrpoBC, we find that rplJp alone is sufficient for high-level expression of these genes.The genes for the subunits of Escherichia coli RNA polymerase are cotranscribed with ribosomal protein genes. The genes for ,B and 13' (rpoB and rpoC) are cotranscribed with at least the L10 and L7/12 ribosomal protein genes (rplL and rplJ, respectively) (26,33,42), whereas the a gene (rpoA) is part of an operon containing four ribosomal protein genes (23). More recently, it has been shown that the gene for a (rpoD) is cotranscribed with the S21 gene (rpsU) and the gene for DNA primase (dnaG) (10).The transcription pattern of the rplKAJLrpoBC gene cluster located at 90 min on the E. coli chromosome is complicated. Initial experiments demonstrated that the genes for the 1B and 1' subunits of RNA polymerase are cotranscribed with at least the rplJ and rplL ribosomal protein genes from a promoter upstream of rplJ (rpUp) (26,33,42). A variety of experiments subsequently suggested that there are also two internal promoters within rplJLrpoBC, one preceding rplL (rplLp) and the other before rpoB (rpoBp) (20,25,26,28,33). In addition, a transcriptional attenuator (atn) was located between rplL and rpoB and accounts for the lower frequency of rpoBC transcription compared with that of rplJL (5,6,14,26). A promoter responsible for the cotranscription of rplK and rplA was identified upstream of rplK (rplKp) (2,15,26,43). Recent S1 nuclease mapping of in vivo transcripts suggests that transcription initiated at rplKp is not necessarily terminated after rplA, but can read through into the rplJL genes (9).To further understand the regulation of this complex operon, we have directly compared the relative effect of these different promoters and the attenuator on rplKAJLrpoBC transcription. These transcriptional regulatory sites were fused, both separately and in combination, to the lacZ gene carried on a lambd...

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“…Thus operation of tl is unlikely to be disturbed by the translation of the upstream mRNA regions. One possible explanation for the high efficiency of rpotl in pHRll would implicate the foreign promoter, as discussed in the accompanying paper (35).…”

Section: Discussionmentioning

confidence: 99%