. Importance of the alpha-subunit of RNA polymerase in UP element recognition .
ID: 88937 • Letter: #
Question
. Importance of the alpha-subunit of RNA polymerase in UP element recognition. Gourse and colleagues performed in vitro transcription on plasmids containing the promoter indicated at top of each gel. They placed the promoters between 100 and 200 nt upstream of a transcription terminator to produce a transcript of defined size. After the reaction, they subjected the labeled transcripts to gel electrophoresis and detected them by autoradiography. The promoters were as follows: -88 contained Wild-type sequence throughout the region between positions -88 and +1; SUB contained an irrelevant sequence instead of the UP element between positions -59 and -41; -41 lacked the UP element upstream of position -41 and had vector sequence instead; lacUV5 is a lac promoter without an UP element; vector indicates a plasmid with no promoter inserted. The positions of transcripts from the rrnB P1 and lacUV5 promoters, as well as an RNA (RNA-1) transcribed from some other gene in the plasmid, are indicated at left. RNAP at top indicated the RNA polymerase used, as follows (a) Wild-type polymerase used throughout. (b) Alpha-235 polymerase (missing the 94 C-terminal amino acids of the alfa-subunit) used throughout. (c) Wild-type (WT) polymerase or R265C polymerase (with cysteine substituted for arginine 265) used, as indicated. Source: Ross et al., A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science 262 (26 Nov 1993) p. 1408.
What are the dark bands at the lacUV5, rrnB P1 and RNA 1 lines? (are they DNA or RNA, and how do you know?
What are wild-type, alpha-235 and R265C?
What part of the RNA polymerase binds to this UP element? How do you know?
Draw the -88, -41 and SUB promoters (like the drawing in question #5), indicating in your drawing how each of these constructs differs from one another?
RNAP lacUV5 rrnB P1 RNA-1 WT Wild-type O-235 R265C WT R265C WT R265 lac lac -88 SUB-88 SUB lacUV5 -88 SUB-41 UV5 vector -88 SUBS -41 UV5 vector lacUV5 rrnB P1 RNA-1 1 2 3 4 5 6 7 8 9 10 1 2 3 CLAAAS 7 8 9 10 4 5 6 1 2 3 4 5 6 78 910 11121314Explanation / Answer
1). Ans : The dark bands at the lacUV5, rrnB P1 and RNA 1 lines, they are the RNA
2). Ans : In given date Alpha -235 polymerase missing 94 C terminal amino acids. Alpha subunit of C Terminal region is very important for UP elements recognitions, and post transcriptional modifications. Thus, Alpha 235 is mutant one than R235C
3). Ans: Alpha subunit of C terminal domain interacts with UP elements because without UP elements infarction transcription doesn’t take place. Up elements are very important transcriptional regulators.
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