Downstream DNA sequence effects on transcription elongation. Allosteric binding of nucleoside triphosphates facilitates translocation via a ratchet motion.
The ability of RNA polymerase (RNAP) to adopt multiple conformations is central to transcriptional regulation. In previous work, we demonstrated that RNAP can exist in an unactivated state that catalyzes synthesis slowly and an activated state that catalyzes synthesis rapidly, with the transition from the unactivated to the activated state being induced by the templated NTP binding to an allosteric site on the RNAP. In this work, we investigate the effects of downstream DNA sequences on the kinetics of single nucleotide incorporation. We demonstrate that changing the identity of the DNA base 1 bp downstream (+2) from the site of incorporation (+1) can regulate the catalytic activity of RNAP. Combining these data with sequence and structural analyses and molecular modeling, we identify the streptolydigin-binding region (Escherichia coli beta residues 543-546), which lies across from the downstream DNA, as the putative allosteric NTP binding site. We present a structural model in which the NTP binds to the streptolydigin loop and upon pairing with the +1 DNA base in the unactivated state or the +2 DNA base in the activated state facilitates translocation via a ratchet motion. This model provides an alternative mechanism for pausing as well as a structural explanation not only for our kinetic data but also for data from elongation studies on yeast RNAP II.
Holmes
Shannon F
SF
Department of Chemistry, the University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Erie
Dorothy A
DA
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D016963:Thermus thermophilus / Q000201:enzymology; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D012321:DNA-Directed RNA Polymerases