Transcription

Question 1

Tell me about rRNA

Answer 1

• comprises 80% of cellular RNA
• transcribed by RNAPI
• are 5S, 5.8S, 18S, and 28S subunits of ribosome

Question 2

Template-product relationship b.w. DNA and mRNA

Answer 2

RNA product looks like the coding strand (not template)

Question 3

RNAP Prokaryotic Holoenzyme

Answer 3

• 2 Alphas, 2 Betas, 1 omega subunits + sigma factor

- several different sigma subunits bind different promoter sequences

Question 4

Transcription Initiation in Prokaryotes

Answer 4

• primary pt of regulation in prokaryotic transc.
• sigma subunit binds -10 (Pribnow) and -35 regions
• DNA gyrase resolves + supercoiling
• TopoI resolves - supercoiling
• no primer needed
• when RNAP starts, sigma factor is left behind

Question 5

Rho Dependent vs. Rho Independent Termination

Answer 5

• Rho protein binds specific RNA sequence as hexamer and contacts RNAP, causing dissociation
• Independent: RNA hairpins generated by palindromic sequences followed by U rich sequences cause RNAP to fall off.

Question 6

Operator in Prokaryotic Transcription Regulation

Answer 6

• usually overlaps promoter

- negative regulatory element that binds repressor protein, which causes RNA to not bind promoter

Question 7

Rifampicin

Answer 7

• binds beta subunit of RNAP and inhibits initiation

- treats tuberculosis

Question 8

Actinomycin

Answer 8

• binds DNA template, phenoxazone ring intercalates b.w. Adjacent GC bps
• blocks elongation
• also interferes with eukaryotes, making it cancer treatment

Question 9

Eukaryotic Promoter Elements

Answer 9

• 25 TATA box, the nucleation site
• promoter proximal elements and enhancers are more upstream
  - PPEs are closer, binding sites for TFs of housekeeping genes. SP1 is a well categorized activator.
  - enhancers can be upstream, downstream, or even within a gene

Question 10

Tata Binding Protein

Answer 10

Part of TFIID

-binds TATA box, causes bend in DNA, allowing TFIIB to bind

Question 11

TFIIB

Answer 11

Binds bent promoter DNA/TFIID

Allows RNAPII + TFIIF to assemble

Question 12

TFIIE and TFIIH

Answer 12

Bind to DNAPII during initiation to help promoter melting

Question 13

5’ Cap

Answer 13

• 7-methylguanosine covalently linked to 5’ end of mRNA via 5’–>5’ triphosphate linkage
• not encoded by DNA template
• added to mRNA shortly after initiation
• recognized by translation machinery to facilitate protein synthesis

Question 14

Poly-A Tail

Answer 14

• happens during termination
• specified by DNA seuqnce
• endonucleolytic cleavage of nascent transcript
• addition of 100-250 residue poly-a tail

Question 15

Steroid Hormone Receptors (structure and mechanisms of action)

Answer 15

• have hormone binding and DNA binding domains
• upon hormone binding, conformational change to allow for DNA binding and to create transcriptional activation domain that binds coactivator complex

2 cases:
Soluble in cytosol
Upon binding hormone, exposure of NLS
Enters nucleus, acts on DNA

Receptor is already in nucleus
Hormone binding either stimulates receptor-DNA binding or induces conformational change in receptor-DNA complex

BOTH REACT WITH COACTIVATOR COMPLEX TO STIMULATE RNA POL II

Question 16

Steroid Response Elements

Answer 16

• enhancers for steroid responsive genes

- palindromic

Question 17

Mechanisms of Transcriptional Activation

Answer 17

• some recruit or stabilize RNAPII/GTP to core promoter

- some clear a path by altering chromatin DNA template

Question 18

Coactivators

Answer 18

2 Classes of Chromatin Modifiers

• catalyzes covalent modification of N-termini of histone tails
• HATs (N termini of H3 and H4) and HDACs
• some methlate, phosphorylate, ubiquitinate
• ATP dependent nucleosome remodeling complexes
• do not catalyze histone modification

Question 19

MMTV

Answer 19

Mouse mammary tumor virus
DNA virus that causes breast epithelial cell carcinoma
Genome includes sequence identical to estrogen enhancer
Integration of MMTV into human genome leads to cancer
Integrates adjacent to wnt-1-proto-oncogene, placing the regulator of cell proliferation under control of estrogens

Question 20

Tamoxifen

Answer 20

Antagonist of estrogen receptor

Fails to induce the conformational change in estrogen receptor that is required for subsequent coactivator binding

Question 21

TFIIH

Answer 21

Plays important role in coupling transcription with DNA damage repair
Altered TFIIH leads to XP, TTD, and CS

Question 22

Alpha-Amanitin

Answer 22

• binds tightly and specifically to RNAPII
• used to distinguish b.w. RNAPII from I and II
• inhibits elongation

Question 23

When does splicing occur?

Answer 23

-either co or post-transcriptionally, but always before translation

Question 24

Mechanism of pre-mRNA splicing

Answer 24

Factors That Contribute to Splicing Fidelity

• base sequence of intron begins with GU and ends with AG
• consensus sequences are found at 5’ and 3’ ends of introns
• branch sequence is found 20-50 nt away from 3’ end of intron
• catalyzed by spliceosome, assembled denovo from SNURPs on precursor RNA that is ATP dependent
• NO ATP is needed for actual transesterification

2 transesterification rxns

• cleavage of PDE bond between exon 1 and intron 1
  - rxn uses 2’OH of branch pt A to form 2’-5’ PDE bond
• 3’-OH of exon 1 cleaves PDE bond bw the intron and exon2
• exon 1 is now joined to exon 2 and intron is released as lariat

Question 25

Splicing errors related to B-Thal

Answer 25

- pt mutation in intron | - create longer or shorter forms of the B-globin

Question 26

SR Proteins

Answer 26

Bind RNA to define exons

Question 27

Factors that chill on CTD of RNAPII

Answer 27

Splicing factors, Cap adders, Poly-A tail proteins (endonuclease and poly-A bps)

Question 28

Proteins on mature mRNAs

Answer 28

Cap and tail proteins, SR proteins on exons Ready by nuclear pore complex

Question 29

Exosome

Answer 29

Degrades RNA debris | Central channel is lined with 3'-->5' exonucleases

Question 30

Lac Operon Control Elements

Answer 30

CAP binding site, upstream of promoter which binds to CAP-cAMP (cAMP binds to CAP in low [Glucose]) THIS CAUSES BENDING IN DNA-->INCREASED AFFINITY FOR RNAPII-->TRANSCRIPTION Operator: downstream of promoter, binds to repressor When repressor binds lactose in low [Glucose], repressor can no longer bind operator.