Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase.
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.
Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, State University of New York Health Science Center, Brooklyn, New York 11203-2098, USA.
Kaiyu
K
Ma
Dmitri
D
Temiakov
Manli
M
Jiang
Michael
M
Anikin
William T
WT
McAllister
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D014764:Viral Proteins
D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases