J. Biol. Chem., Vol. 265, Issue 35, 21430-21432, Dec, 1990
Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity and a nonprocessive T7 RNAP mutant
R Sousa, YJ Chung, WT McAllister, BC Wang and EM Lafer
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Two RNA polymerases homologous to bacteriophage T7 RNA polymerase,
bacteriophage Sp6 and T3 RNA polymerases, were screened for crystallization
under conditions identical or similar to those reported for the growth of
large single crystals of T7 RNA polymerase (Sousa, R., Rose, J. P., Chung,
Y. J., Lafer, E. M., and Wang, B.-C. (1989) Proteins 5, 266; Sousa, R.,
Lafer, E. M., and Wang, B.-C. (1990) J. Crystal Growth, in press; Sousa,
R., and Lafer, E. M. (1990) Methods 1, in press). A number of mutant T7
RNAPs were also screened under these conditions as were three chimeric RNA
polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal
sequences. One chimeric polymerase and two mutant polymerases crystallized
readily under T7 RNAP crystallization conditions. The chimeric polymerase
crystallized in a space group different from T7 RNA polymerase: orthorombic
with unit cell parameters a = 75 A, b = 98 A, c = 159 A; space group
P2(1)2(1)2(1) and 4 molecules/unit cell. This chimeric enzyme exhibits T3
promoter specificity and will make it possible to investigate how
structural differences between the T3 and T7 RNA polymerase promoter
recognition domains determine their different promoter specificities. One
of the mutant polymerases successfully crystallized was an enzyme which can
carry out promoter recognition and abortive transcription but cannot carry
out processive transcription. Its structure may provide information on the
nature of the conformational changes undergone by T7 RNAP in the
abortive-processive switch. Crystals of the second mutant T7 RNA polymerase
were unsuitable for x-ray analysis.
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