Determinants of the stability of transcription elongation complexes: interactions of the nascent RNA with the DNA template and the RNA polymerase.
We use a synthetic "primed bubble-duplex" model elongation complex developed previously to examine certain structural and thermodynamic features of the transcription elongation complex of Escherichia coli. The nucleic acid framework of this model complex consists of a linear base-paired DNA molecule with a central "bubble" of non-complementary nucleotide residues, together with a single-stranded RNA molecule that is complementary (at its 3'-end) to three to 12 nucleotide residues of one of the DNA strands within the bubble. RNA polymerase is added to this framework in trans, and on addition of rNTPs the resulting complex can elongate the 3'-end of the RNA primer in a template-dependent manner with functional properties that are indistinguishable from those of a "natural" promoter-initiated transcription elongation complex operating under the same conditions. In this study we use this model system to show that the formation of a stable elongation complex at any particular template position can be treated as an equilibrium process, and that semi-quantitative dissociation constants can be estimated for the complex by using a gel band-shift assay to monitor the binding of the RNA oligomer to the complex. We then show that the formation of a stable complex depends on the presence of a complementary RNA-DNA hybrid that is at least 9 bp in length, and in addition that several nucleotide residues of non-complementary RNA located upstream of the RNA-DNA hybrid bind strongly to the putative single-stranded RNA binding site of the polymerase and significantly enhance the stability of the resulting elongation complex. Finally, we demonstrate that the measured stabilities of the model constructs in which the length of the RNA-DNA hybrid is varied correlate well with the transcriptional processivity of the functioning complex that results when rNTPs are added. These findings are discussed in the context of related studies of both model systems and natural elongation complexes. The general concepts that emerge are used to define some central structural and functional features of the transcription complex.
Wilson
K S
KS
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR, 97403, USA.
Conant
C R
CR
von Hippel
P H
PH
D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D012329:RNA, Bacterial / Q000096:biosynthesis*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases