Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.
The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.
Severinov
K
K
Rockefeller University, New York, NY 10021, USA.
Mustaev
A
A
Severinova
E
E
Bass
I
I
Kashlev
M
M
Landick
R
R
Nikiforov
V
V
Goldfarb
A
A
Darst
S A
SA
D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases