SCIENCE AT THE EDGE SEMINAR

QB/GEDD

 

Friday, May 28 at 11:30am

Room 1400 Biomedical and Physical Sciences Bldg.

Refreshments at 11:15

 

 

Ronald S. Johnson

Brody School of Medicine at East Carolina University, Greenville, NC

 

 

 

Rapid Kinetic Analysis of Transcription Elongation as Catalyzed by RNA Polymerase

 

 

The elongation complex of RNA polymerase (RNAP) is capable of rapidly and efficiently carrying out the process of transcription. The nucleotide addition cycle plays an important role in this process. However, it is poorly defined. We have performed a series of stopped-flow and quench-flow kinetic studies along with footprinting studies in order to better define this cycle. Incorporation of each nucleotide during transcription results in the generation of pyrophosphate (PP_i) at the active site. Our kinetic studies indicate that the intrinsic rate of PP_i release is too slow to account for the rapid rate of elongation. Only in the presence of the next correct nucleotide for incorporation is the rate of PP_i release rapid and consistent with the overall rate of elongation. Footprinting studies with various elongation complexes indicate that RNAP in the presence of PP_i is predominantly in the pre-translocated state. These data led us to postulate that the next correct nucleotide for incorporation binds at a putative pre-insertion site where it basepairs with the template strand and elicits a conformational change in RNAP leading to rapid PP_i release. To further define the nucleotide addition cycle, we used the intrinsic protein fluorescence of RNAP to monitor NTP binding with subsequent nucleotide incorporation in stopped-flow kinetic studies. The rate of the conformational change associated with this process is comparable to the rate of PP_i release in the presence of the next correct nucleotide for incorporation and suggests that these two processes are linked. In studies comparing the results between NTP and NPCPP (a nonhydrolyzable nucleotide analog that cannot be incorporated into RNA), a similarly fast conformational change in RNAP was observed with the NPCPP analog. This indicates that the conformational change occurs prior to chemistry and provides supporting evidence for the postulated mechanism for the nucleotide addition cycle involving the rapid release of PP_i.

 

 

Helen Geiger

Administrative Assistant

Quantitative Biology Initiative/

Gene Expression in Development & Disease

Michigan State University

502B Biochemistry Building

East Lansing, MI   48824

Phone:  (517) 432-9895

Fax:  (517) 353-9334

E-mail: [log in to unmask]

Web: http://qbmi.msu.edu

http://www.bch.msu.edu/GEDD/index.htm