In vivo characterization of tus gene expression in Escherichia coli

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.The chromosomes of Escherichia coli and Bacillus subtilis, and some plasmids like R6K, initiate replication from specific origins and the replication forks moving either bi-or unidirectionally, are arrested at sequence-specific replication termini (1-3). Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4, 5), plants (6), Xenopus (7), and human (8).The replication termini are sequence-specific and bind to cognate terminator proteins, and the protein-DNA complex arrests replication forks in an orientation-dependent manner (9, 10). DnaB (11,12,19). Detailed biochemical and biophysical analyses have been made possible in both the systems by the determination of the crystal structures of RTP at 2.6 Å (21) and of Tus-Ter () complex at 2.7 Å (22). The DNA binding (23), dimer-dimer interaction (24), and DnaB interaction domains (25) of RTP have been determined by genetic and biochemical analyses that used the crystal structure as a guide. Similarly, the DNA binding domain of Tus has been determined with the help of crystal structure and genetic analysis (22).The impact of transcription on the initiation (26, 27) and elongation stages of DNA replication have been reported (28,29). We have reported previously that Tus and RTP can block RNA chain elongation catalyzed by several prokaryotic RN...

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“…arrest of transcriptional elongation has also been implicated in the autoregulation of Tus protein of E. coli (47).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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“…Lp-=--i,.,.nd....,.e-pe-n:d~~,· t·r~~Scriplion A transcript starting from the PI promoter is arrested in vivo at the .2 terminus that also serves as an operator of the Ius gene and binds Ter (Tus) protein to transcriptionally autoregulate Ter (Tus) synthesis (54,55).…”

Section: The Impact Of Transcription On Sequence-specific Fork Arrestmentioning

confidence: 99%

Termination of DNA Replication in Prokaryotic Chromosomes

Bastia

Manna

Sahoo

1997

Genetic Engineering

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“…While replication arrest is nonessential, the importance of this process is made apparent by the similarity of the components of the E. coli and S. typhimurium systems (Roecklein et al 1991) and the existence of different but functionally analogous systems found in an increasing number of other organisms. That termination of DNA replication and replication arrest are not equivalent has been demonstrated most clearly in strains lacking either the gene for the DNA-binding terminator protein or the termination sites themselves.…”

Section: Introductionmentioning

confidence: 99%

Progress in Botany

1997

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In vivo characterization of tus gene expression in Escherichia coli

Cited by 23 publications

References 28 publications

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Termination of DNA Replication in Prokaryotic Chromosomes

Progress in Botany

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