Transcription and its control

Question 1

Differences b/w Transcription and DNA replication (4)

Answer 1

1. only 1 DNA strand is transcribed (RNA is identical to non-template/coding strand)
2. RNA primer not required
3. All of the DNA in a cell is replicated by DNA pol but only a fraction of genome transcribed
4. different enzymes (DNA pol are faster and more accurate)

Question 2

Similarities b/w Transcription and DNA replication (3)

Answer 2

1. incorporation of nucleoside triphosphates
2. DNA template directed
3. template directed extension of nucleic acid chains in 5’ to 3’ direction

Question 3

Prokaryotic Promoter Recognition

Answer 3

• conserved sequences in promoters recognized by E.coli
• promoter region is -70 to +30
• nucleotides near -35 or -10 regions in contact w/ pol (predominantly the sigma unit)
• spacing b/w -35 and -10 regions critical for maintenance of DNA protein contacts
• promoter mutations that increase/decrease sigma binding are clustered in -10 and -35 regions

Question 4

Prokaryotes-The transcription Elongation Complex

Answer 4

• transcription brings about significant changes in supercoiling of DNA (dealt w/ by topoisomerase)
• protein-RNA, protein-DNA-RNA, Protein-DNA contacts are extensive
• 17 bp of DNA is unwound w/ Tc bubble
• polymerase can pause, translocate backwards and cleave the 3’ end of nascent RNA (error correction? improving the Tc of difficult regions of template?)

Question 5

Prokaryotes-Rho factor dependent termination

Answer 5

• RNA-DNA helicase Tc termination factor, nucleoside triphosphatase activity activated upon binding to polynucleotides
• rho recognizes CA rich region (rut site) near the 3’ end of the transcript , where RNA pol has paused
• rho moves toward the 3’ end of the transcript by unwinding the RNA-DNA duplex and pulling it away from the RNA pol
• RNA-DNA unwinding is ATP dependent

Question 6

Prokaryotes-Rho factor independent termination

Answer 6

• A rich segment on template strand (>3 nt but usually more)
• GC rich region upstream of As, capable of forming hairpin by self complementarity
• hairpin formed (like above), pol pauses
• A-U base pairs disrupted, weakly bound oligo (U) tail released

Question 7

DNA footprinting

Answer 7

a technique used to identify/study regions of DNA bound by proteins

• solution of radioactively labelled (at 1 end) DNA fragments
• treat w/ DNase, no cuts made in the region where RNA pol is bound
• isolate labeled DNA fragments and denature
• separate by polyacrylamide gel, visualize bands in x ray film

Question 8

Prokaryotes-mapping Tc start points

Answer 8

Primer Extension Method
-end label the restriction fragment internal to the promoter
-hybridization of probe and mRNAs
-Rt
-run samples on polyacrylamide gel, autoradiography
Key points:
-know exactly where the probe binds the mRNA
-RT rxn will go until it reaches the end of template
-length of labeled cDNA tells you position of Tc initiation

Question 9

Transcription and its control in eukaryotic cells

Answer 9

• 3 different multisubunit RNA pol complexes (I, II, III)

- many additional Tc factors needed to recognize promoter and initiate Tc

Question 10

Transcription Factors

Answer 10

• have DNA binding domains and regulatory domains used to interact w/ RNA pol and Tc proteins
• some Tc factors common to all 3 RNA pol complexes
• others are complex specific

Question 11

RNA pol II elongation complex-Similarities to prokaryotes (3)

Answer 11

• issues w/ supercoiling
• similar size and structure of Tc bubble
• protein-RNA, protein-DNA, protein-protein interactions

Question 12

Nucleolus

Answer 12

• site of rRNA Tc (RNA pol I) and ribosome biogenesis
• contains DNA, RNA, protein
• built around the regions of chromosomes that have rRNA genes