Dataset Information

Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.

ABSTRACT: By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.

SUBMITTER: Inoue N

PROVIDER: S-EPMC207244 | biostudies-other | 1991 Feb

REPOSITORIES: biostudies-other

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Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.

Inoue N N Uchida H H

Journal of bacteriology 19910201 3

By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascen ...[more]

PMID: 1846858

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Project description:Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch repair (MMR)-defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base pair substitutions or small indels (i.e., insertion and deletions of ≤4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hot spots for indels. No additional orientation bias in mutation frequencies occurred when MMR- strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus codirectional to replication. In addition, the data revealed a strong correlation of the frequency of base pair substitutions with gene length but no correlation with gene expression levels. IMPORTANCE Because DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. However, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair-defective Escherichia coli strains, we found that across the genome there are no significant differences in mutation frequencies between genes oriented codirectionally and those oriented head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for small insertions and deletions in head-on-oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hot spots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and codirectionally to replication.

Dataset Information

Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.

Publications

Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.

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