Lecture theme 4 The Process of Transcription

Question 1

Transcription by RNA polymerase III.

Answer 1

 Polymerase III promoters can be located upstream or downstream of the transcribed region

 5S rRNA gene promoter is located downstream within the 5S gene

Question 2

Strategy for identifying the location of promoters:

deletion mutation analysis

Answer 2

If the region that occurs upstream of gene is deleted and results in no transcription, promoter is located upstream of +1.

If the region that occurs upstream of gene is deleted and transcription still occurs, the promoter must be located downstream of +1.

Question 3

Technology for identifying the exact basepairs where a protein binds to element: DNase I footprinting assay.

Answer 3

• end label DNA fragment
• mix with cell extract (contains proteins)
• digest with DNase I using conditions where all possible fragments are generated (lengths differ by single nt)
• separate fragments by PAGE
• detect by autoradiography

 Region bound to protein is protected against digestion, i.e. no bands where protein has bound

Question 4

Transcription by RNA polymerase II.

Answer 4

 Transcription initiation: at TATA box ~ 30 bp upstream of +1

 TATA is first DNA target site for assembly of the basal transcription complex.

1-TFIID binds to TATA box.
2-TFIID-DNA recruit RNA poly TFIIB binds.
3-TFIIB recruits RNA POLYMERASE II with TFIIH Kinase.
4-RNA POLYMERASE is phosphorylated and transcription iniates.
5-kinase and polymerase move along the DNA.

Question 5

Elongation of transcription.

Answer 5

 RNA pol II first phosphorylated by TFIIH at S5; is essential to initiate transcription
 RNA pol II pauses after producing a short 20-30 nt transcript
 elongation requires further phosphorylation of RNA pol II
 by recruitment of pTEF-b kinase; enzyme phosphorylates pol II at S2

Question 6

Termination of transcription.

Answer 6

• initial RNA transcript is cleaved downstream of the conserved polyadenylation signal AAUAAA
• polyA tail is then added to the free 3’ end

Question 7

Transcription by RNA polymerases.

Answer 7

RNA polymerase I (mainly rRNA)

RNA polymerase II (all mRNAs)

RNA polymerase III (all tRNA and small RNAs)