T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide

The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production...

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Bibliographic Details
Published in:Biochemistry (Easton) 1989-04, Vol.28 (7), p.2760-2762
Main Authors: Martin, Craig T, Coleman, Joseph E
Format: Article
Language:English
Subjects:
Base Sequence
DNA-Directed RNA Polymerases - metabolism
Escherichia coli - enzymology
Kinetics
Molecular Sequence Data
Substrate Specificity
T-Phages - enzymology
Transcription, Genetic
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Summary:The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production of the five-base runoff transcript GGACU. In particular, Km for the interaction between enzyme and template under saturating levels of all four nucleotide triphosphates was shown to be approximately 0.02 microM. We now show that the corresponding Km and Vmax for initiation on a similar template coding for the runoff transcript GACU are the same as for the earlier study (Km = 0.02 microM; kcat = 40-50 min-1). This new template allows the measurement Km for association of the initial nucleotide GTP with enzyme or with the enzyme-DNA complex. The results show that KGTPm (0.60 mM) is somewhat higher than earlier approximations of Km for addition of elongating GTP during the later phase of processive elongation. As expected, the (initiating) Km for the GTP analogue ITP (KITPm) is increased (by about 2-fold), presumably as a result of weakened Watson-Crick base pairing. However, comparison of Km values for the GTP analogues GMP and guanosine shows little effect on substitution of the 5'-triphosphate by monophosphate or by a hydroxyl, respectively. This result suggests that a single active site has been evolutionarily adapted to accept from the 5' end of a waiting nucleotide both a 5'-triphosphate at initiation and a 5'-monophosphate ester (RNA) during elongation.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00433a002