Y. Miwa et al., STRUCTURAL AND FUNCTIONAL DISSECTIONS OF TRANSCRIPTION TERMINATION FACTOR-RHO BY RANDOM MUTAGENESIS, Journal of Molecular Biology, 254(5), 1995, pp. 815-837
Transcription termination factor rho from Escherichia coli is a homohe
xamer of 419 amino acid subunits and catalyzes an ATP-dependent releas
e of nascent RNA transcripts. A rho monomer has three distinct domains
functioning independently at the first approximation: the amino-termi
nal one quarter containing a primary RNA-binding site, the central 270
-amino acids region constituting an ATP-binding domain with homologies
to F-1-ATPase, and the carboxy-terminal remainder with unknown functi
on(s). To further delineate the structural and functional organization
s of rho protein, we undertook its random mutagenesis using error-pron
e polymerase chain reactions with the carboxy-terminal 100-amino acid
region chosen as the initial target. From 14 mutants identified, rho p
rotein was purified and characterized in vitro. Of these, 11 mutants a
re defective in termination in vivo and show decreased activities in v
arious partial functions examined: ATP binding; RNA binding; and ATPas
e activities dependent on three cofactors with decreasing efficacies,
poly(C), hero RNA and poly(U). A few of them are also affected in the
putative secondary RNA-binding site that is functionally coupled to AT
P hydrolysis. By contrast, the three other mutants are hyperactive in
termination, poly(U)-dependent ATPase activity, and RNA interaction at
the primary site. Ln these properties, the hyper-terminating mutants
strikingly resemble the ''super rho'' mutant formerly found in the ami
no-terminal domain. Taken together, these findings indicate that the c
arboxy-terminal region plays a pivotal role in functionally coupling t
he RNA and ATP-binding domains, plausibly by acting as an interface fo
r their interaction within or across individual subunits. In light of
the reported X-ray crystallographic structure of R-ATPase, we propose
a model for the tertiary and quaternary structure of rho that is consi
stent with the observed mutational effects as well as a number of stru
ctural and functional properties characteristic of rho. (C) 1995 Acade
mic Press Limited