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Slide-8 - #8 Prokaryotic transcription (I) 1. 2. 3. 4. 5....

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#8 Prokaryotic transcription (I) 1. The transcription reaction 2. Bacterial RNA polymerase 3. The promoter and the sigma factor 4. Transcription initiation and elongation 5. Transcription termination (Pages 135-145, 150-161 are not covered)
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1. The transcription reaction -All cellular RNAs are synthesized by the transcription process. -Transcription is a DNA-dependent RNA polymerization reaction catalyzed by the RNA polymerase -Only one of the two strands of DNA is copied into RNA, transcription process is asymmetric. 3’ ATGCATGCATGC 5’ RNA 5’ UACGUACGUACG 3’ DNA 5’ TACGTACGTACG 3’ RNA polymerase 3’ 5’ 5’
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1. RNA elongates in the 5’ 3’ direction. 2. Transcription produces single strand RNA that has the identical sequence (except T to U) with the so called transcribed strand of DNA. Transcribed strand : with the same sequence as the RNA product. Template strand : with the sequence complementary to the RNA Upstream : toward 5' of the transcribed strand Downstream : toward 3' of transcribed strand Direction of Transcription 5’ 3’ 3’ 5’ upstream downstream
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How fast is transcription? 1. Transcription rate is about 1kb/min. So, it takes only ~4 min to transcribe a 4kb gene. 2. Cells may need thousands of mRNA of the same gene to make protein synthesis fast enough, so it often starts the next round of transcription before the previous one finishes (as shown). RNA DNA
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2. Bacterial RNA Polymerase RNA polymerase was purified from E. coli (1969). It is a multiple subunit enzyme with the following composition: Alpha ( α ), 40 kD Beta ( β ), 150 kD Beta' ( β ’), 160 kD Sigma factor ( σ 70 ), 70 kD Omega ( ω ) 10 kD not found in the SDS-PAGE shown was later discovered (not essential) Core enzyme: 2 α + β + β ' Holoenzyme: 2 α + β + β '+ σ 70 + ω The textbook description of this gel (p127) is wrong, so use the labels on this slide! σ * α β β ' σ Core enzyme ( α +2 β ) α +2 β + σ holoenzyme contaminant σ C B A GG (original label)
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were done in the 1960’s to establish the function of each subunit of this enzyme. For example, the results from at least three experiments demonstrated the important functions of sigma factor, which was originally purified in the holoenzyme but not included in the core (or minimum) enzyme. What is the function of Sigma factor? Experiment 1: In an in vitro transcription experiment, the core enzyme (2 α + β + β ') has much lower activity using an intact DNA than using a nicked DNA as the template, no such difference was observed for the holoenzyme. Moreover, holoenzyme transcribes only specific sequences but the core enzyme was indiscriminative. Sigma determines specificity.
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Slide-8 - #8 Prokaryotic transcription (I) 1. 2. 3. 4. 5....

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