Department of Molecular Biology and Biochemistry
Waksman Institute of Microbiology
190 Frelinghuysen Rd., Room 224
Piscataway, NJ 08855-0759
Genetic and biochemical analysis of RNA polymerases from E. coli and yeast, site-directed modification of proteins
Transcription, the first step, and a major regulatory checkpoint of gene expression is carried out by DNA-dependent RNA polymerases. RNA polymerase alone or in complex with regulatory factors is central to all steps of transcription. Defective transcription is the cause of aberrant growth and development and may result in malignant transformation. Our long-term scientific goal is to understand transcription mechanism and regulation in molecular detail.
One project in this laboratory concerns domain organization of the two largest subunits (ß' and ß) of RNA polymerase from Escherichia coli. Our goal is to use a combination of biochemical, genetic, chemical and structural approaches to bridge the gap between the primary RNA polymerase sequence, available functional data, and the three-dimensional model of RNA polymerase. We have showed that 25% of the RNA polymerase ß subunit sequence is dispensable and could be deleted without affecting basic function. A principal result was the demonstration that dispensable regions could be involved in interactions with transcription factors. We also demonstrated that both ß' and ß can be physically split without preventing RNA polymerase assembly and function. These results showed that RNA polymerase is a highly modular enzymes and opened several new avenues of research which are currently being pursued. The assembly-competent subunit fragments are being used to investigate intersubunit interactions during RNA polymerase assembly. Split RNA polymerases are also being used to map chemical crosslinks between RNA polymerase and derivatized nascent RNA or DNA template.
This work is now also being extended to RNA polymerase I (pol I) from yeast. We use a unique genetic system that makes pol I dispensable for cell viability to uncover structure-functional relationships of this enzyme. In addition. we use pol I as a vehicle to assemble in vivo chimeric RNA polymerases,harboring domain swaps between pol I and pol II and pol III.
Finally, in collaboration with a chemistry lab we are designing a general method of site-specific, chemical modification of proteins. We developed a very important technique that allows us to incorporate fluorescent and crosslinkable labels within ca. 50 C-terminal amino acids of a protein. Our approach involves an in vitro ligation of the smaller, chemically synthesized C-terminal fragment of a protein to the larger, recombinant N-terminal fragment which is genetically fused to protein self-splicing element intein. At conditions favoring intein excision and in the presence of the C-terminal fragment containing N-terminal cysteine efficient ligation of the N and the C-terminal segments is achieved. Our immediate plans are to use this system to systematically study protein-protein and protein-nucleic acids interactions in transcription complexes.