Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus.
RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (alpha(2), beta, beta', omega and sigma(70)) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 A resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P3(2), with unit-cell parameters a = b = 236.35, c = 249.04 A, and have perfect twinning along the threefold axis. A complete data set at 3 A resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the sigma(70) subunit is now in progress.
Genomic Sciences Center (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Marina N
MN
Vassylyeva
Jookyung
J
Lee
Shun Ichi
SI
Sekine
Oleg
O
Laptenko
Seiki
S
Kuramitsu
Takehiko
T
Shibata
Yorinao
Y
Inoue
Sergei
S
Borukhov
Dmitry G
DG
Vassylyev
Shigeyuki
S
Yokoyama
D003460:Crystallization; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D016963:Thermus thermophilus / Q000201:enzymology*
D012321:DNA-Directed RNA Polymerases