Transcription is the major control
point of gene expression and RNA polymerase (RNAP) is the central
enzyme of transcription. The purpose of our research is to elucidate
the physical basis of transcription and its regulation by defining
the physical interactions between RNAP, DNA, RNA, and associated
regulatory factors. These interactions are best elucidated through
structural studies of the macromolecules and assemblies involved.
Most information about the fundamental mechanism of transcription
comes from studies of E. coli RNAP, making it an excellent
model. This is especially true because of the conservation of
RNAP structure and mechanism from bacteria to man. Because of
the large size and complexity, these studies require a combination
of methods. Electron microscopy (EM) is used
to determine low resolution (10-25 Å) structures of intact
assemblies. To obtain high-resolution information, we use X-ray
crystallography to study RNAP subunit domains. This work relies
heavily on biochemical studies to define and characterize, structurally
and functionally, appropriate subdomains for study. Finally, the
results of EM and X-ray studies will be combined to build a picture
of the complete RNAP assembly.
EM Studies of E. coli
RNAP Core Enzyme. Progress to date on structure determination
of multisubunit RNAPs has been due to the study of two-dimensional
(2-D) crystals by EM. RNAPs form 2-D crystals on lipid layers
and 3-D structures of E. coli RNAP holoenzyme
(containing the promoter specificity factor 
)
and yeast RNAP II were determined
(Darst et al, 1989,
Nature 340, pp. 730-732 and
Darst et al.,1991, Cell
66, pp 121-128). We have now determined
the structure of E. coli RNAP core enzyme
(lacking ) at about 20 Å resolution.
Interestingly, the E. coli core RNAP structure bears striking
resemblance to the yeast RNAP II structure.
This may define an elongating conformation" of the enzyme,
as both the E. coli core and yeast RNAP II enzymes are
capable of RNA chain elongation but not specific promoter recognition
in the absence of additional factors.
Structural and Functional Studies
of E. coli RNAP Subunit Domains. Work in our laboratory
and many others has demonstrated that the E. coli RNAP
subunits (
2ßß´
)
are each comprised of smaller subdomains. We are working to identify,
then structurally and functionally characterize, the subdomains
of all the subunits. Recent work has been focused on the
and ß subunits.
To identify the subdomains of ,
we use limited proteolytic digestion to identify protease-resistant
fragments. We have now completely mapped the trypsin degradation
pathway of and are heavily engaged
in structural and functional studies of each of the domains.
A powerful in vitro functional approach has proven effective
in investigating the subdomain architecture of the ß subunit.
Using this approach, we have shown that ß split at defined
sites into as many as three separate peptides can still assemble
into functional enzyme with the other RNAP subunits in vitro.
These results indicate that the ß subunit is comprised of
at least three independent domains and delineates a strategy that
we are using to define further the domain architecture of the
ß and ß´ subunits.
Structural and Functional Studies
of Transcript Cleavage Factors from E. coli. Transcription
elongation factors stimulate the activity of RNAPs by increasing
the overall elongation rate and the completion of RNA chains.
One group of such factors, which includes E. coli GreA,
GreB, and eukaryotic SII (TFIIS), acts by inducing hydrolytic
cleavage of the transcript within the RNAP, followed by release
of the 3´-terminal fragment. We have determined the structure
of GreA by X-ray crystallography (in collaboration with S. Borukhov
and A. Goldfarb, Public Health Research Institute). The structure
contains a very unusual intermolecular, antiparallel -helical
coiled-coil dimer which extends into solution. Cross-linking studies
show that a site near the tip of the coiled-coil "finger"
plays a direct role in the transcript cleavage reaction by contacting
the 3´-end of the transcript. Current structural and functional
studies are focused towards elucidating the transcript cleavage
mechanism.
