Transcription_10.16

Spt4, Spt5, Spt6

Pausing involves “backtracking”, in which the polymerase moves back 2-4 nucleotides on the template, unwinding the 3’ end of the transcript from the template bubble

true

• Arrest occurs by backtracking of 7-14 nucleotides
• Backward movement misaligns the 3' end of the nascent RNA with the RNAPII active site, thereby prohibiting continued RNA synthesis

• TFIIS
• It does this by inducing internal cleavage of the displaced nucleotides and creating a new paired 3' end that is properly aligned

GTF TFIIF reduces pausing and arrest and may protect nascent RNA from TFIIS-induced cleavage

30%

Pausing is especially common in genes that belong to various signal-responsive pathways

• DRB sensitivity-inducing factor (DSIF=Spt4+Spt5) and negative elongation factor (NELF) facilitate Pol II pausing in the promoter-proximal region
• TFIIS associates with the paused polymerase and stimulates the intrinsic RNA-cleavage activity of Pol II to create a new RNA 3'-OH in the Pol II active site after backtracking of the polymerase
• 

DSIF, NELF and Ser2 on the Pol II CTD -> productive elongation is stimulated

• Model 1: polyA signal leads to changes in composition of RNAP
• Model 2: mRNA is cleaved at polyA signal, generating new 5’-end that is rapidly degraded (torpedo model)

• mRNA is cleaved at polyA signal, generating new 5’-end that is rapidly degraded (torpedo model)
• 

promoter-selectivity factor SL1 consists of the TATA-binding protein (TBP) and three or more TBP-associated factors (TAFI) bind to the core promoter

The homodimeric DNA-binding protein upstream binding factor (UBF) recognizes upstream control element (UCE)

Transcription intermediary factor IA (TIF-IA) binds to RNAPI and SL1 and assists in RNAP recruitment

Two other essential factors, TIF-IC and general transcription factor TFIIH (not shown), are also thought to be involved in RNAPI-mediated transcription initiation

• SL1 (consisting of TPB, core promoter adn 3+ TBP factors)
• TIFIA
• TIFIC
• TFIIH

Most RNAPIII-transcribed genes have internal promoters within the transcribed region, which are recognized by the large, five-subunit factor TFIIIC

• TFIIIC recognizes internal promoters for the genes to be transcribed and then recruits TFIIIB
• TFIIIB helps open the promoter and then recruits RNAPIII

TFIIIA

The difference between activators (or repressors) and co-activators (or co-repressors) is lack of DNA-binding domain in the latter

the Gal4 recognition element

• Transcription assays
• (Ultrabithorax) causess extra pair of wings from a third thoraxic segment

biochemical purification

• DNA molecules containing tandems of the TF binding site are synthesized and coupled to a solid support resin to create a sequence-specific affinity column
• Sequence-specific TFs are purified in a two-step procedure
• First many different DNA binding proteins are eluted and separated from total cell proteins
• Then a sequence specific column matrix has the DNA binding proteins from step 1 run over it, and the column collects any specific transcription factors

acetylate histone proteins, weakening the association of DNA with histones and making the DNA more accessible to transcription => transcription is induced

deacetylate histone proteins, strengthening the association of DNA with histones and making the DNA less accessible to transcription => transcription is repressed

• DNA binding domain
• Transcription activation domain

• ligand binding
• phosphorylation
• interaction with other transcription factors
• interaction with co-regulatory proteins

Ligand binding that can influence where a TF is located within a cell (cytoplasm versus nucleus) and whether the transcription factor is in an active state and capable of binding DNA or other cofactors (e.g., steroid hormone receptors)

Phosphorylation that is a prerequisite of the TF binding to its specific promoter element (e.g., STAT proteins)

Interaction with other TFs (e.g., homo- or hetero-dimerization) or with co-regulatory proteins that results in a conformational change and, hence, sequence-specific DNA recognition

• Acidic domains (e.g., in Gal4: 11/49 aa in domain are acidic)
• Glutamine-rich domains (e.g., in Sp1: 39/143 aa are Gln in that domain)
• Proline-rich domains (e.g., in CTF/NF-I: 16/100 aa are Pro)

• Helix-turn-helix (HTH)
• Zinc-containing modules (zinc fingers)
• Basic region leucine zipper (bZIP) motifs
• Basic helix-loop-helix (bHLH) motifs

Dimerization of TFs doubles the number of contacts and quadruples affinity for binding sites (i.e., the square of the number of contacts)

Most activators have to work at very low concentrations, so high affinity of TFs for their target sites is critical!

dimerize

Many classes of DNA binding proteins contain an a helix in their DNA binding domain that contacts the major groove in the double helix

by the hydrogen bonding patterns between amino acid side chains of the TF and nitrogenous bases in the DNA

present in both prokaryotes and eukaryotes

The N-terminal helix is a “bracing” helix, whereas the C-terminal helix is the “recognition” helix; both make contact with DNA

The recognition helix interacts with the major groove

The recognition helix projects into the DNA major groove at various angles and forms hydrogen bonds and Van der Waals interactions with exposed bases

as monomers

Homeobox is a 180 bp DNA sequence found both in vertebrates (including mammals) and invertebrates and it encodes a 60 aa-long HTH DNA-binding motif

• homeotic transformations
• loss-of-function mutations in the regulatory region of the Antennapedia gene result in the development of the second leg pair into ectopic antennae, whereas gain-of-function alleles convert antenna into ectopic legs

TFIIIA

only a few bases of DNA