Lecture 13 Euk Trxn 2 - RNAP subunits and regulatory...

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RNAP subunits and regulatory sequences http://genome.imim.es/~dfarre/courses/transcription.jpg 1 Tuesday, October 11, 2011
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Isolation of RNA polymerases RNAP I, RNAP II, RNAP III RNA polymerase transcribes: rRNA (RNAP I), mRNA (RNAP II), tRNA/ small rRNA (RNAP III) Last Time 2 Tuesday, October 11, 2011
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RNAP subunits and regulatory sequences Subunit structure of RNAP II (identiFed using epitope tagging technique) DNA interaction with RNAP II SigniFcance of RPB1 phosphorylation Eukaryotic promoter elements (overview) Determination of theTATA box function in SV40 using S1 mapping 3 Tuesday, October 11, 2011
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Learning Outcomes 1. Discuss the experimental design identifying the subunits of RNAP II (Epitope Tagging). Be able to apply this technique to a new problem. 2. Compare/ contrast prokaryotic and eukaryotic RNAP subunits in terms of structure and function. 3. Describe how RNAP II is modiFed to move from initiation to elongation phase. 4. Identify RNAP II promoter elements 5. Discuss and be able to apply the experimental technique (S1 Mapping) identifying the function of the TATA box. By the end of this lecture, you should be able to: 4 Tuesday, October 11, 2011
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All RNAPs have a similar subunit structure 5 Tuesday, October 11, 2011
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Subunit structure of all RNAPs All have 10-14 subunits Subunits range from 10kDa to 220 kDa All have 2 very large subunits and several smaller ones Several of the smaller subunits are common (shared?) to all 3 RNAPs 6 Tuesday, October 11, 2011
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A closer look at Yeast RNAP II 12 subunits 10 subunits are essential, & two more are required only under certain conditions The 2 large subunits (genes Rpb1 and Rpb2 ) are structurally and functionally homologous to β and β ( E.coli RNAP) Rpb1 targeted by α -amanitin 7 Tuesday, October 11, 2011
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Experimental Question: What are the subunits of RNAPII? 8 Tuesday, October 11, 2011
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Experimental technique - epitope tagging Method to identify (and isolate) subunits of large protein complexes. ...among other uses! (It’s kinda like goin’ Fshin’!) http://www.sunrealtync.com/Fishing-1-tight%20crop.jpg 9 Tuesday, October 11, 2011
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Experimental technique - epitope tagging http://www.sunrealtync.com/Fishing-1-tight%20crop.jpg Solid support (pole) ANTIBODY specifc to TAG (hook) epitope TAG (fsh mouth) protein oF interest (fsh)
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This note was uploaded on 03/29/2012 for the course BIO 359K taught by Professor Mcclelland during the Spring '08 term at University of Texas at Austin.

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Lecture 13 Euk Trxn 2 - RNAP subunits and regulatory...

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