Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Our laboratory studies transcript initiationand elongation by RNA polymerase II.We are currently focusing on (i) theprocess by which RNA polymerase II beginstranscription and clears the promoter; that is, thetransition from initiation to the transcriptelongation phase of RNA synthesis, (ii) themolecular mechanisms involved in the elongationprocess, and (iii) the effects of chromatinstructure on transcript elongation. All of theseaspects of transcription are important checkpointsin the regulation of gene expression. Theultimate goal of these studies is a more comprehensivepicture of transcription through itsaccurate duplication in test-tube systems.We have recently found that repetitivetemplate sequences in the initially transcribedregion can allow the nascent RNA to slipupstream and reassociate with the template,leading to the synthesis of an RNA longer thanpredicted by the template sequence (Pal andLuse, 2002). This observation has proved to bean important tool in allowing us to understandthe structural transitions within the RNApolymerase that must occur so that the initial,unstable transcription complex can successfullypass into the processive transcript elongationphase (Pal and Luse, submitted; Pal and Luse, inpreparation).Several major projects are under way onpromoter clearance and transcript elongationmechanisms. The first is an extension of ourexperiments (Samkurashvili and Luse, 1998) inwhich we explored the linkage betweentranslocation of RNA polymerase II along thetemplate and transcriptional arrest. We showedthat the polymerase tends to slide back along thetemplate, and in some cases to arrest, until about25 bonds have been made. Surprisingly, the fullymature form of the transcription complex doesnot emerge until the nascent RNA is about 45bases long. We have recently found that thisbehavior is a complex function of bothtranscript length and template sequence (Pal etal., 2001). We have also found that thetransition into the fully processive form of thetranscription complex is reversible (Újvári andLuse, 2002). Dr. Újvári, is currently extendingher recent observations to investigate interactionsbetween the transcript and the proteins ofthe transcription complex that stabilize thepolymerase in the elongation-committed state.We are also studying the sequence requirementsfor arrest by polymerase II (Hawryluk and Luse,submitted). Our results should help to explainthe known tendency of RNA polymerase II topause about 25-40 bases into the transcriptionunit for many eukaryotic genes.The Department of Molecular BiologyRNA Polymerase II Function Evaluated inTranscription Initiation, Checkpoints forTranscript ElongationOur other major project concerns theability of RNA polymerase II to elongate nascentRNAs on nucleosomal templates. We have shownthat nucleosomes assembled from highly purifiedhistones form an essentially absolute barrier toelongation by RNA polymerase II (Chang andLuse, J. Biol. Chem. 1997;272:23427) and thatprotein factors can partially relieve this barrier(Orphanides et al., 1998). We are currentlyfocusing on the effects of nucleosome structureon transcript elongation by RNA polymerase IIand, in a collaborative study with the laboratoryof Vasily Studitsky, on mechanistic aspects of thenucleosomal block to transcript elongation.Donal S. Luse, Ph.D.THE LUSELABORATORYRESEARCH ASSOCIATESMahadeb Pal, Ph.D.Subramaniam Sanker, Ph.D.POSTDOCTORAL FELLOWSLouise Steele, Ph.D.Andrea Újvári, Ph.D.TECHNICAL ASSOCIATESusan W. LuseCOLLABORATORVasily M. Studitsky, Ph.D. 11Dept. of Biochemistry andMolecular Biology, Wayne StateUniv., Sch. of Med., Detroit, MIOrphanides, G., LeRoy, G., Chang, C.-H., Luse, D.S., and D. Reinberg (1998) FACT, a factorthat facilitates transcript elongation through nucleosomes. Cell 92:105-116.Samkurashvili, I.. and D.S. Luse (1998) Structural changes in the RNA polymerase II transcriptioncomplex during the transition from initiation to elongation. Mol. Cell. Biol. 18:5343-5354.Pal, M., McKean, D., and D.S. Luse (2001) Promoter clearance by RNA polymerase II is anextended, multistep process strongly affected by sequence. Mol. Cell. Biol. 21:5815-5825.Pal, M., and D.S. Luse (2002) Strong natural pausing by RNA polymerase II within 10 basesof transcription start may result in repeated slippage and reextension of the nascent RNA.Mol. Cell. Biol. 22:30-40.Újvári, A., Pal, M., and D.S. Luse (2002) RNA polymerase II transcription complexes maybecome arrested if the nascent RNA is shortened to less than 50 nucleotides. J. Biol.Chem. 277:32527-32537.Pal, M., and D.S. Luse (2003) The initiation-elongation transition: lateral mobility of RNA inRNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23. Proc. Natl.Acad. Sci. USA 100:5700-5705.107