The molecular mechanisms of transcription termination and pausing in the Escherichia coli threonine operon regulatory region

To study the mechanism of transcription termination at a rho-independent terminator, homoduplex and heteroduplex templates that contained mutations in the G + C-rich region of dyad symmetry in the Escherichia coli thr operon attenuator were constructed. Assays of these templates in an in vitro transcription system showed that the DNA sequence of the template strand determines the transcription termination efficiency. There was no indication that the potential complementary base pairs between the non-transcribed DNA strand and the nascent RNA in the G + C-rich region are involved in transcription termination. The results argue against models where DNA-DNA pairing or RNA pairing with the non-transcribed DNA strand in a bridged intermediate are important for transcription termination.The thr attenuator contains a run of six consecutive A residues immediately preceding the G + C-rich dyad symmetry which can potentially form rA-rU base pairs with the distal U residues. The possible contributions of the rA and rU interactions to transcription termination at the thr attenuator were also examined. A set of templates bearing base substitutions in the A and T stretches of the thr attenuator which were constructed by oligonucleotide-directed site-specific mutagenesis. The in vitro and in vivo results indicate that the A stretch is not critical in determining the termination efficiency of the thr attenuator.E. coli RNA polymerase pauses at distinct sites distal to a region of dyad symmetry which encodes the 1:2 hairpin in the thr leader RNA. These pause sites are closely-spaced to each other and occur at positions G$\sp{112}$, G$\sp{114}$ and A$\sp{116}$ of the thr leader RNA. Structural analyses of the paused complex showed that a region of approximately 35 bp on both strands of the DNA template was protected by RNA polymerase from DNase I and hydroxyl radical cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. In addition, the nature of interactions between RNA polymerase and DNA template remain unchanged in the presence of NusA protein.A set of mutants which contained altered sequences upstream and downstream of the transcriptional pause sites were constructed to examine the mechanism of transcriptional pausing in the thr leader region. The results showed that there is no strict correlation between the predicted stability of the RNA hairpin and the half-life of the paused complex and that the sequences where pausing normally occurs or downstream of the pause sites can affect pausing. In addition, in vitro transcription studies with DNA templates containing successively shorter thymidine tracts downstream of the G + C-rich dyad symmetry of the thr attenuator showed that the intact G + C-rich region of the thr attenuator is not sufficient to make RNA polymerase pause.U of I OnlyETDs are only available to UIUC Users without author permissio