Transcription Elongation Complex Stability

Abstract

Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi-and single subunit RNA polymerases have evolved an 8 -10-base pair RNA-DNA hybrid as a part of a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4-base pair hybrid can nevertheless be more stable than those with the normal 8-base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.RNA polymerases are inherently processive in that, unlike distributive DNA polymerases, an incompletely synthesized RNA cannot be further extended by the binding and action of a second polymerase. Unlike DNA polymerases, an RNA polymerase also must maintain a limited length nascent (hybrid) duplex, dissociating the 5Ј end of the transcript as nucleotides are added to the 3Ј end, whereas the short RNA-DNA hybrid must resist the collapse of the transiently melted DNA bubble. Thus the evolution of complex stability in an RNA polymerase is fundamentally different from that of a DNA polymerase. Why does an RNA polymerase elongation complex maintain an RNA-DNA hybrid of ϳ8 -10 bases, regardless of the size of the polymerase? A reasonable answer to this question might propose that this is the minimum length required for thermodynamic stability of an RNA-DNA hybrid, because hybrids of this length have stabilities ranging from 2 to 10 kcal/mol in solution (1).Several factors have been considered to contribute to the stability of elongation complexes, including interactions between the polymerase and the nucleic acids (DNA and RNA), and the interactions between individual nucleic acid strands (2-4). These interactions need to be sufficiently strong to prevent the premature release of transcripts over thousands of bases yet labile enough to ensure rapid elongation (50 -250 base pairs/s). It is expected that the interactions during the elongation phase are non-sequence-specific.It is now apparent that there are two major families of RNA polymerase: 1) the multi-subunit family comprising both the eukaryotic and bacterial RNA polymerases and 2) the "single subunit" RNA polymerase family comprising both the T7 family of RNA polymerases and the (two subunit) mitochondrial/ chloroplast RNA polymerases. That all of these complexes contain an 8 -10-base hybrid in the elongation complex (5-10) suggests that this length is dictated by the process rather than by enzyme specifics.In the study of elongation complex stability in vitro, one can artificially omit one of the substrate nucleoside triphosphates, leading to a halting of elongation at the first occurrence of the omitted nucleotide (11,12). The stability ...

New Insights into Transcription Fidelity: Thermal Stability of Non-Canonical Structures in Template DNA Regulates Transcriptional Arrest, Pause, and Slippage

“…It was reported that polymerase backtracking appears to pass through a paused, transcriptionally inactive intermediate state [28], [49]. The molecular basis for backtracking is not clear, but it is correlated either with mispairing[50], [51], [52] or very weak pairing of the 3′ end of the transcript and the template [49], [53]. Current models require that the RNA-DNA hybrid at the point of arrest be very weak to initiate upstream translocation [50].…”

The Fidelity of Transcription

“…The mutation might facilitate the 3Ј RNA shifting during slippage. Indeed, recent report on bacteriophage T7 RNA polymerase indicates that the 3Ј RNA extrusion during backtracking may constitute a part of the slippage mechanism (36). In addition, a short distance 1-2-bp backtracking from the end of 11A tract induces pausing of RNA polymerase without moving the enzyme away from the homopolymeric sequence.…”

The Presence of an RNA:DNA Hybrid That Is Prone to Slippage Promotes Termination by T7 RNA Polymerase

“…The extensive protein:nucleic acid interactions observed in the RNA:DNA hybrid cavity, and/or the topological intertwining of the nucleic acid components, may therefore stabilize an otherwise unstable hybrid configuration 17,18,40 .…”