Effects of Solution Conditions on the Steady State
Kinetics of Initiation of Transcription by T7 RNA Polymerase
Maribeth Maslak and Craig T. Martin
Biochemistry, 33, 6918-6924, 1994.
The T7 family of DNA-dependent RNA polymerases presents
an ideal model system for the study of fundamental
aspects of transcription. The small size of the promoter
allows a variety of studies based on simple steady
state kinetics in the synthesis of a 5 base runoff
transcript. This assay can be used to characterize
the effects on the initiation of transcription of simple
modifications to potential protein or DNA specificity
contacts. In the current work, in vitro conditions
for this assay have been identified which optimize
the apparent Km for the interaction between enzyme
and the promoter DNA. The addition to the reaction
of 0.05% Tween-20 and the substitution of 10 mM NaCl
by 100 mM potassium glutamate not only improves the
quality of the kinetic assays, but also decreases Km
by about an order of magnitude (strengthening the interaction
between polymerase and its promoter). As observed for
DNA binding in other systems, the parameter Km increases
substantially with increasing [NaCl], but the salt
dependence is shifted to higher concentrations as a
function of [KGlu]. Thermal denaturation of the protein,
monitored by circular dichroism spectroscopy, confirms
the effects of salt and supports a model in which Cl-
and other anions compete for phosphate binding sites
on the protein. Finally, while Km is highly dependent
on [NaCl], the measured kcat is relatively insensitive
to salt. These data indicate that the parameters Km
and kcat reflect changes, respectively, in promoter
binding and in a rate limiting step or steps leading
to the initiation of transcription.
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Questions? CMartin@Chem.UMass.edu