Transient state kinetics of transcription elongation by T7 RNA polymerase.
The single subunit DNA-dependent RNA polymerase (RNAP) from bacteriophage T7 catalyzes both promoter-dependent transcription initiation and promoter-independent elongation. Using a promoter-free substrate, we have dissected the kinetic pathway of single nucleotide incorporation during elongation. We show that T7 RNAP undergoes a slow conformational change (0.01-0.03 s(-1)) to form an elongation competent complex with the promoter-free substrate (dissociation constant (Kd) of 96 nM). The complex binds to a correct NTP (Kd of 80 microM) and incorporates the nucleoside monophosphate (NMP) into RNA primer very efficiently (220 s(-1) at 25 degrees C). An overall free energy change (-5.5 kcal/mol) and internal free energy change (-3.7 kcal/mol) of single NMP incorporation was calculated from the measured equilibrium constants. In the presence of inorganic pyrophosphate (PPi), the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 s(-1) with PPi Kd of 1.2 mM. Several experiments were designed to investigate the rate-limiting step in the pathway of single nucleotide addition. Acid-quench and pulse-chase kinetics indicated that an isomerization step before chemistry is rate-limiting. The very similar rate constants of sequential incorporation of two nucleotides indicated that the steps after chemistry are fast. Based on available data, we propose that the preinsertion to insertion isomerization of NTP observed in the crystallographic studies of T7 RNAP is a likely candidate for the rate-limiting step. The studies here provide a kinetic framework to investigate structure-function and fidelity of RNA synthesis and to further explore the role of the conformational change in nucleotide selection during RNA synthesis.
Anand
Vasanti Subramanian
VS
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Patel
Smita S
SS
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D003101:Collodion / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism / Q000502:physiology*; D011756:Diphosphates / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism / Q000502:physiology*
D011756:Diphosphates; D004983:Ethanolamines; D014764:Viral Proteins; D003101:Collodion; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C007369:2-diethylaminoethanol