Abstract
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.
Highlights
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]
We have reported previously that Tus and replication terminator protein (RTP) can block RNA chain elongation catalyzed by several prokaryotic RNA polymerases in a polar mode and that the passage of an RNA transcript that invades the terminus from the permissive direction causes functional inactivation of the replication terminus [30]
We endeavored to address four important questions regarding the interplay between transcription and replication termination. (i) What is the eventual fate of RNA polymerase (RNAP) when it reaches the Tus-Ter () complex that is positioned in blocking orientation? Is the RNAP arrested or does it dissociate from the DNA leading to authentic termination of transcription? (ii) Does the passage of transcription through the replication terminus cause the dissociation of the Tus protein from the DNA-protein complex abrogating replication fork arrest or is it caused by a transcription-mediated conformational change of the Tus-Ter () comerase chain reaction; DTT, dithiothreitol; TE, Tris1⁄7HCl plus EDTA. 2 A
Summary
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]. We linearized pET22b- DNA carrying the TerB (see Fig. 1) site of E. coli in the orientation that impedes replication forks [11, 12], and initiated transcription from a T7 promoter by T7 RNAP in the presence of 0-, 2-, 4-, or 8-fold molar excess of Tus over DNA substrate (100 fmol of DNA and 0, ϳ7, 14, and 28 ng of Tus).
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