The +2 NTP binding drives open complex formation in T7 RNA polymerase.
Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation. Transient state kinetic studies show that the open complex, ED(o), is formed via an intermediate ED(c), and the conversion of ED(c) to ED(o) occurs with an unfavorable equilibrium constant. In the presence of the initiating NTP that base-pairs with the template at position +2, the process of open complex formation is nearly complete. Our studies reveal that the nucleotide that drives open complex formation needs to be a triphosphate and to be correctly base-paired with the template. These results indicate that the melted template DNA in the open complex is positioned to bind the +2 NTP. The addition of +1 NTP alone does not stabilize the open complex; nor is it required for +2 NTP binding. However, there appears to be cooperativity in initiating NTP binding in that the binding of +2 NTP facilitates +1 NTP binding. The dissection of the initiation pathway provides insights into how open complex formation steps that are sensitive to the promoter sequence upstream from the initiation start site modulate the affinity of initiating NTPs and allow transcription initiation to be regulated by initiating NTP concentration.
Stano
Natalie M
NM
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Levin
Mikhail K
MK
Patel
Smita S
SS
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012265:Ribonucleotides / Q000378:metabolism*; D013816:Thermodynamics; D014158:Transcription, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D012265:Ribonucleotides; D014764:Viral Proteins; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium