Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling.
A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.
Grachev
M A
MA
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Academy of Sciences of USSR.
Lukhtanov
E A
EA
Mustaev
A A
AA
Zaychikov
E F
EF
Abdukayumov
M N
MN
Rabinov
I V
IV
Richter
V I
VI
Skoblov
Y S
YS
Chistyakov
P G
PG
D000345:Affinity Labels; D001345:Autoradiography; D001665:Binding Sites; D003488:Cyanogen Bromide; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006868:Hydrolysis; D008239:Lysine; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000345:Affinity Labels; D010446:Peptide Fragments; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D008239:Lysine; D003488:Cyanogen Bromide