[Show abstract][Hide abstract] ABSTRACT: Unlike DNA polymerases, RNA polymerases (RNAPs) must displace the nascent product from the template and restore the DNA to duplex form after passage of the transcription complex. To accomplish this, RNAPs establish a locally denatured "bubble" that encloses a short RNA:DNA hybrid. As the polymerase advances along the template, the RNA is displaced at the trailing edge of the bubble and the two DNA strands are reannealed. Structural analyses have revealed a number of elements that are likely to be involved in this process in T7 RNAP. In this work, we used genetic and biochemical methods to explore the roles of these elements during the transition from an initiation complex to an elongation complex. The results indicate that the transition is a multistep process and reveal a critical role for the nontemplate strand of the DNA.
[Show abstract][Hide abstract] ABSTRACT: This chapter analyzes the probing of the organization of transcription complexes using photoreactive 4-Thio-substituted analogs of uracil and thymidine. The use of 4-thio-substituted UTP or dTTP analogs provides a powerful method to probe the organization of transcription complexes. These analogs may be placed at defined positions in the transcript or the DNA, and on UV activation form cross-links to nearby regions of the protein with high efficiency. A number of analogs have been employed in these studies. Subsequent mapping of the site of the cross-link by peptide mapping provides important information concerning the organization of the complex and the trajectory of the nucleic acid components over the surface of the protein. The preparation of transcription complexes with specifically positioned photoreactive analogs is experimentally designed. The design of the template is dictated by the purpose of the experiment and the position in the template or product that is being analyzed. The exact strategy for mapping the site of the cross-link depends on the suitability of various chemical or enzymatic cleavage reagents with the particular protein being examined.
Methods in Enzymology 02/2003; 371:133-43. DOI:10.1016/S0076-6879(03)71009-X · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid approximately 9 bp upstream from the active site (at -9) it interacts with a region within the specificity loop (residues 744-750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172-191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination.
[Show abstract][Hide abstract] ABSTRACT: We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between −5 and −2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or “scrunch” the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.