Our lab is currently engaged in two major projects:

1. Structure-function of RNAP and its binary and ternary complexes with DNA template and RNA transcript;

2. The function and the molecular mechanism of action of prokaryotic transcript cleavage factors GreA and GreB.

1. RNA Polymerase (RNAP) is the key enzyme of the transcriptional process. Its basic structure-functional features are highly conserved among all living organisms. In bacteria, the catalytically active multisubunit core enzyme (subunit composition: a2bb'w with the total molecular mass of ~400 kDa) joins s factor to form the holoenzyme (a2bb'ws), which is capable of specific promoter recognition and efficient initiation of transcription. The transcription cycle of RNAP consists of several steps including initiation, elongation and termination. The biochemical activities of this multifunctional enzyme include recognition and specific binding of promoter DNA, melting of the DNA double helix, binding of the nucleotide substrates and formation of the first phosphodiester bond, RNA chain extension and translocation along the DNA template, recognition of signals for pausing, RNA termination and antitermination. In addition to RNA polymerization, RNAP is also capable of RNA hydrolysis by exo- and endonucleolytic reactions. Most of the RNAPÕs functional activities are subject to regulation by a large variety of external factors.

The last five years mark the breakthrough in our understanding of structural and functional organization of RNAP. The 3D-structural information was obtained for bacterial and yeast core RNAPs, s and a domains, and complexes of RNAP with nucleic acids. The latest major advance was the high-resolution X-ray structures of bacterial holoenzyme from extreme thermophiles, Thermus thermophilus (Tth) and Thermus aquaticus (Taq), and the structure of Taq holoenzyme-promoter DNA complex. Together with an array of genetic, biochemical, and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of transcription cycle. However, many aspects of the transcription process, including the molecular mechanisms of basic biochemical reactions catalyzed by RNAP are still poorly understood.

To address these questions, we need to see more high-resolution structures of RNAP complexes with DNA, RNA, and nucleotide substrates with and without other transcription factors, which reflect the complexity of every step of the transcription cycle. Also, further biochemical and genetic analysis will be required to complement and refine the structural data. To this end, in our lab we are developing new approaches to prepare stable binary (ÒopenÓ and ÒclosedÓ) RNAP-promoter DNA complexes, and ternary elongation complexes (RNAP-DNA-RNA), that will be amenable for X-ray crystallographic analysis. As in our previous studies (Fig. 1, Publications 3, 4), we plan to use in our experiments RNAP core and holoenzyme isolated from Thermus thermophilus.

	Fig. 1 Thermus thermophilus RNAP holoenzyme at 2.6� resolution Vassylyev, et al (2002) Nature , 417, 712-719

2. Prokaryotic transcript cleavage factors GreA and GreB affect the efficiency of transcription elongation in vitro and in vivo by stimulating the intrinsic nucleolytic activity of RNAP. The cleavage of nascent RNA is an evolutionarily conserved function among all multisubunit RNAPs. It occurs in backtracked ternary complexes (TCs), typically 2-18 bases upstream from the 3'-terminus, followed by dissociation of the 3'-proximal RNA fragment and restart of transcription from newly generated 3'-terminus. GreA induces hydrolysis of mostly di- and trinucleotides, whereas GreB induces cleavage of fragments of various lengths, depending on the extent of RNAP backtracking. Biochemical studies of Escherichia coli Gre factors indicate that they are not nucleases but RNAP co-factors, which activate the same catalytic center that is involved in both RNA synthesis and RNA hydrolysis reactions.

 

The biological role of factor-induced endonucleolytic reaction includes: (i) the enhancement of transcription fidelity, by helping RNAP excise misincorporated nucleotides; (ii) suppression of transcriptional pausing and arrest by reactivation of RNAP during reversible and irreversible backtracking; and (iii) stimulation of RNAP promoter escape and transition from initiation to elongation stage of transcription by helping the catalytic center to re-engage with nascent RNA 3'-terminus during abortive synthesis. The biological significance of Gre factors is underscored by the fact that Gre-homologs have been found in more than 60 bacterial organisms, including Mycoplasma genitalium, an organism with the smallest known genome, and several extremophiles, such as Thermus thermophilus; and Dinoccocus radioduran.

All members of Gre family are homologous polypeptides of ~160 amino acids. They have similar structural organization and surface charge distribution, and are made of two domains: an N-terminal extended coiled-coil domain (NTD) and a C-terminal globular domain (CTD). The NTD is responsible for the induction of type-specific nucleolytic activity by Gre factors as well as readthrough and antiarrest activities whereas CTD is responsible for the high affinity binding of Gre to RNAP. One of the distinct structural features of most Gre factors is the basic patch, a cluster of positively charged residues present on the side of the protein surface that is presumed to face RNAP in TC. Basic patch residues are responsible for Gre interaction with nascent RNA in TC and are required for efficient antiarrest activity. The exact molecular mechanism by which Gre factors activate the catalytic center of RNAP and induce the RNA cleavage in TC is unknown. The current model of Gre action involves the following three essential steps: the binding of Gre-CTD to RNAP near the secondary channel opening; the interaction of Gre-NTDÕs basic patch with the 3Õ-terminal portion of the transcript extruding from the secondary channel in backtracked TC, and the activation of RNAPÕs endonucleolytic activity via residue(s)-specific interaction between RNAP and Gre-NTD.

The broad goal of this project is to understand the mechanism of action and the structure-function relationships of GreA and GreB. We plan to carry out four types of experiments: (i) To identify functionally important localities of Gre factors, we use oligonucleotide-directed random mutagenesis to introduce single amino acid substitutions of those residues in Gre proteins that are not involved in intramolecular interactions and site-directed mutagenesis to introduce single and multiple amino acid substitutions in the conserved loop, and in the interface between the N- and C-terminal domains. The mutants will be characterized by specific transcription assays in vivo and in vitro. (ii) To elucidate interactions between Gre and other components of the TC, we will identify mutations in the b and bÕ subunits of RNAP that suppress the lethal phenotypes of dominant negative mutant Gre factors or overproduction of wt Gre. The mutant RNAPs will be purified and characterized by functional assays in vitro. (iii) Interactions between Gre proteins and RNA polymerase are investigated using Fe2+-induced hydroxyl radical footprinting and mapping, and specific cysteine-directed protein-protein photochemical cross-linking. (iv) To obtain 3D-structural information, we plan to co-crystallize Thermus thermophilus Gre factors with RNAP core and holoenzyme and subject them to X-ray crystallographic analysis.

 

SUPPORT

Our work is supported by grant from the National Institutes of Health.

 

PERSONNEL

Jookyung Lee, Ph. D.,
Principal Research Scientist,

Oleg Laptenko,
Research Assistant.

 

SELECTED PUBLICATIONS

1. Koulich, D., Lee, J., Lomakin, I., Nowicka, B., Das, A., Darst, S. A., Normet, K., & Borukhov, S. (2000) The Functional Role of Basic Patch, a Structural Element of Escherichia coli Transcript Cleavage Factors GreA and GreB. J. Biol. Chem., 275, 12789-12798.

2. Borukhov, S. Laptenko, O., & Lee, J. (2001) Transcript cleavage factors GreA and GreB: Functions and mechanism of action. Meth. Enzymol. "Ribonucleases" 342, 64-76.

3. Vassylyeva, M.N., Lee, J., Sekine, S., Laptenko, O., Kuramitsu, S., Shibata,T., Inoue,Y., Borukhov, S., Vassylyev,D.G., and Yokoyama, S. (2002) Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus. Acta Crystallogr. 58, 1497-1500.

4. Vassylyev, D.G., Sekine, S., Lee,J., Laptenko, O., Vassylyeva, M.N., Borukhov,S., and Yokoyama, S. (2002) Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6� resolution. Nature , 417, 712-719.

5. Borukhov, S. & Severinov, K. (2002) Role of the RNA polymerase sigma subunit in transcription initiation. Research in Microbiology. (in press).

6. Borukhov, S & Nudler, E. (2003) RNA polymerase holoenzyme: structure, function, and biological implications. Curr. Opin. Microbiol. (in press)