Chapter 5: Transcription

Question 1

Transcription of gene information

Answer 1

• DNA information into ribonucleic acid (RNA).
• RNAcontains the sugar ribose and uracil.
• RNA polymerase binds to the transcriptional start site in DNA and uses one of the DNA strands as a template for transcription.
• RNA strand grows in a 5’ 3’ direction.
• Template DNA is copied 3’ 5’.

Question 2

RNA polymerase

Answer 2

2 Alpha (assembley of the core) 
2 Beta (Confers ability to bind DNA), 
1 sigma (Allows promoter recognition and binding)

Question 3

Footprinting 5

Answer 3

1) A sequence of DNA bound to a protein (RNA polymerase) is partially digested with DNase.
2) Cleaved positions are recognized by using DNA labeled in one strand at one end.
3) Broken DNA fragments are electrophoresed on a gel separating according to length of fragments.
4) In free DNA, every bond position is broken. But the region covered by RNA polymerase (protein) is protected.
5) Two reaction are run: free DNA (control) + a DNA-protein

Question 4

Mutations identify DNA regions involved in binding

Answer 4

Mutations at -35 consensus sequence reduce the rate of recognition by RNA polymerase.
Mutations at -10 slow unwinding of DNA and initiation of transcription (notice -10 is AT rich).

Question 5

Substitution of sigma factors and regulation of gene expression

Answer 5

Different sigma factors bind to different promoters so different genes are transcribed. The sigma factor responsible for transcription of genes under normal conditions is 70. High temperature rpoH gene produces 32 initiate transcription of heat shock proteins.

Question 6

Elongation of RNA chains

Answer 6

Catalyzed by the RNA polymerase core enzyme.Extension occurs within a locally unwound segment of DNA. RNA polymerase: Unwinds DNA ahead of polymerization site and rewinds behind. The region of base-pairing between growing chain and template is very short. The stability of the transcription complex is maintained by the binding of the RNA polymerase to DNA/RNA.
Holoenzyme: needed for the initiation of transciption

Question 7

Termination of transcription

Answer 7

1) mGC rich stretch followed by a polyA on the template strand. The GC in the RNA pair forming a dsRNA (hairpin loop). polyU
2) rho-dependent termination: Rho binds to the rutsite in RNA. After binding, pulls RNA off the RNA polymerase. rut is close to sequences at which RNA polymerase pauses.

Question 8

Rho-independent termination

Answer 8

RNA polymerase reaches the end of the gene (AAAA). Hairpin-loop forms upstream the open complex. Formation of loop causes polymerase to pause. While pausing the hybrid DNA-RNA hybrid dissociate (weak U:A bonds).

Question 9

Rho-Dependent termination

Answer 9

• rho-dependent termination: Rho binds to the rutsite in RNA. After binding, pulls RNA off the RNA polymerase. rut is close to sequences at which RNA polymerase pauses.
• Rho moves towards 3’ end, displacing the DNA template strand. The displacement weakens the template-transcript interaction, causing dissociation

Question 10

Transcription in Eukaryotes

Answer 10

There are three different RNA polymerases in eukaryotes:
-RNA polymerase I –>most rRNA;
-RNA polymerase II –>mRNA;
-RNA polymerase III –>tRNA, snRNA, 5S rRNA.
All three eukaryotic RNA polymerases require other proteins (transcription factors) to synthesize RNA. Presence of nucleus–>RNA processing + transcription. DNA is organized into chromatin that can block access of RNA polymerase

Question 11

Initiation of transcription in eukaryotes

Answer 11

Transcription factors must bind to a DNA promoter before RNA polymerase binding and transcription.
RNA polymerase II
Conserved promoter sites are:
TATAbox (5’-TATAAAA-3’) centered at position –30.;
CAATbox (GGCCAATCT) centered at position –80.;
GCbox (GGGCGG) centered around -50

Question 12

Three major modifications of transcripts in eukaryotes

Answer 12

Processing:
•Methyl guanosine caps (MG caps) are added to the 5’ end.
•Non-coding regions (introns) are spliced out.
•Poly(A) tails are added to the 3’ (mediated by endonuclease and poly A polyeramse)

All of this phosphorylation of the DTD of RNApol is critical in signalling recruitment of these proteins.