Lecture 20: Eukaryotic Transcription

What happens to RNA Pol II's that allows it to enter the elongation phase?

More phosphates are added to the serine 2 of the heptanucleotide repeat of the CTD

What is the consensus sequence of the CTD for RNA pol IIa?

YS(2)PTS(5)PS(7)

What do these five conformation states of TFIID represent?

The range of mation TBP undergoes during the formation of the full promoter factors

What are trans-acting factors?

The regulatory transcription factors that bind to cis-acting elements. The transcription factors that control the expression of a gene are themselves encoded by genes; regulatory genes that encode transcription factors may be far away from the genes they control.

What does TFIIS do?

They are factors that promote the hydrolysis of backtracked nascent RNA after the misplacement of an incorrect base

What are cis sequence elements?

They are regions (TATA box, CAAT box, GC box) on a specific strand of DNA that is nearby the other regions of a specific gene to recruit trans-acting factors via DNA protein interactions

What is the role of the TAF's?

They bind interact with activator proteins

How do transcription factors exert their effects on transcription?

They influence transcription by regulating the rate at which the assembly of the basal transcriptional apparatus occurs.

How do Actinomycin D, and acridine do?

They intercalate between successive G=C base pairs, but it inhibits transcription elongation in eukaryotes and prokaryotes

What do all eukaryotic RNA polymerases require to bind to a promoter and intiate?

They require transcription factors

What form of Pol II is recruited to promoter?

Unphosphorylated form

Is polyadenylation in bacteria as well?

Yes, but the polyadenylated transcripts in bacteria are targeted for degredation

What are the three regions of the Mediator (in yeast cells)?

Head, middle, and tail

What is TFIIH? (4 things)

1. 9 polypeptides, and a core of 6 subunits. 2. Helicase in both the 5'-3' and 3'-5' direction. 3. involved in NER 4. has 3 subunit Cdk7/MO15 + cyclin H + MAT1 kinase that phosphorylates Ser5 of CTD during transcription initiation

What are the 5 parts of the polyadenylation complex?

1. CPSF: cleavage and polyadenylation specificity factor 2. CStF: cleavage stimulator factor 3. CFI and CFII: cleavage factor I and II 4. PAP: Poly-A polymerase 5. PABPII: Poly-A Binding Protein II

What are the five conformation states of the TFIID? (CExSREn)

1. Canonical 2. Extended 3. Scanning 4. Rearranged 5. Engaged

What are some common protein domains for transcription factors?

1. DNA Binding domains (Helix-turn-helix, leucine zipper motif, helix-loop-helix) 2. Activation domain (interact with basal machinery to increase rate of transcription) 3. Dimerization domain (protein-protein interactions) 4. Ligand binding domain

What are transcription inhibitors useful?

1. Development of antibiotics 2. Facilitate studying of transcription enzyme function. 3. Study the half life of mRNA in question

What happens typically after RNA pol II reaches the end of a gene?

1. It continues to transcribe well past the end. 2. RNA pol II will go through AATAAA sequences on the DNA that is 3' to the gene. 3. The mRNA contains AAUAAA sequence that will be recognized by an endonuclease that cuts 11 to 30 nt downstream of the sequence 4. poly-A polymerase adds up to 200 adenine residues

In order of most sensitive to least, order the RNA pol's that are sensitive to alpha-amanitin?

1. Pol II 2. Pol III 3. Pol I (not sensitive)

Why is it beneficial to have mRNA capping, polyadenylation and splicing occuring while transcription is occuring?

1. Processing factors are at high local conc. when the splice sites and poly(A) signals are being produced. 2. Association of splicing factors with 2 and 5 serine phosphorylated on CTD signals for elongation. (pre-mRNA production is reliant on having the necessary factors there to process it)

Describe the mechanism of action of the polyadenylation complex?

1. RNA is cleaved 10-35 nt 3' of the AAUAAA 2. Poly-A polymerase binds to the newly cleaved 3' end is rapidly polyadenylated 3. PAP adds first 12A res slowly 4. Binding of PAPBII stabilizes the PAP, allowing quicker polyadenylation

What is the binding order of the transcription machinery at the initiation site?

1. TFIID (TBP binds to TATA sequence) 2. TFIIB 3. TFIIF - RNA polymerase 4. TFIIE 5. TFIIH

What are distant-independent elements?

1. regions of DNA that can act up to a maximum of 50kb away from the sequences they regulate. 2. Can be downstream or upstream 3. Can act in either orientation 4. Can elevate the initiation of transcription (enhancers) or reduce the rate (silencers)

What is TFIIA?

2-3 subunit protein that increases the affinity of TBP for DNA. Possible anti-inhibitor

What does BRE, DPE, CTF, CBF, TBP, and TAF stand for?

BRE = TFIIB recognition element DPE = downstream promoter element CTF = CCAAT-binding transcription factor CBF = CCAAT-box binding factor TBP = TATA-box binding protein TAF = TBP associated factors

What dies rifampicin do?

Binds beta-subunit of bacterial RNA polymerase and it blocks promoter clearance (elongation)

What is TFIIF (RAP30/74)?

Binds to RNA pol II. and aids in transcription initiation and elongation

Why can Pol I and Pol III use a small number of ubiquitous TF's while Pol II uses a large variety of specific TF's?

Due to Pol II's role at transcribing mRNA, the utilization of a greater and more specific variety of TF's allows for more intricate regulation.

What are the main products of eukaryotic RNA pol I, II, and III?

I = rRNA (28S, 18S, 5.8S) II = mRNA & small RNAs III = tRNAs, 5S, and small RNAs

How many different RNA Pol are there for eukaryotes?

I, II, and III

What is the rate of transcription of a gene defined by?

In both eukaryotes and eukaryotes, the rate is determined by the sum of positive and negative regulatory influences that are exerted by proteins bound to cis acting elements

What is TFIIB?

It binds to TBP and BRE

What portion of the DNA does the TBP bind?

It binds to the minor groove

What can the deletion of the TATA box cause?

It can result in different transcription start sites without eliminating transcription in general

What is TFIID?

It consists of the TBP and all the associated TAF's.

How is TFIIB similar to the sigma factor?

It contains topological similarities. B ribbon: (similar to sigma 4, flap tip helix domain) B linker (sigma 2) both bind coiled-coil... B core: (sigma 3) B reader:

What does two activities TFIIH complex have?

It has helicase and it is the kinase. It unwinds DNA and phosphorylates the CTD of Pol II, allowing for the elongation complex

What does the Mediator complex do?

It integrates a bunch of physiological and developmental signals and transmits these signals to Pol II. It links the enhancer-bound activators to promoter-bound pol II

What is P-TEFb?

It is a kinase that phosphorylates CTD of pol II, resulting in the release from the pause stage of elongation

What is the Mediator complex?

It is a large multi-subunit protein that allows for the regulation of gene expression through activator-dependent transcription

What is the polyadenylation complex?

It is a protein complex that interacts with the CTD of Pol II after initiation.

What is Sp1?

It is a transcription factor that is required for SV40.

Why is the leucine zipper called as such?

It is due to the presence of leucine residues that causes the binding of two loops. The leucines are like the zippers.

What cells have the mediator complex?

It is found in all eukaryotic cells

What hapens shortly after RNA Pol II begins transcription?

It is phosphorylated on the serine 5 of the heptanucleotide repeat by TFIIH and the 5' cap is placed on the RNA

What happens to the poly-A tail over time?

It is shortened gradually.

What is the CTD?

It is the C-terminal domain of RNA polymerase (YSPTSPS) that is repeated up to 60 times (depends on species). These residues can be modifed, say by phosphorylation, to influence the function of eRNAP II

What does CTD mediate?

It mediates the placement of the 5' cap, splicing machinery, and the polymerase that adds the poly-A tail.

Besides activator-dependent transcription, what else does the mediator complex do?

It stimulates activator-independent (basal) transcription and can be a co-repressor to cause transcriptional silencing. It can influence all stages of the transcription cycle

How can an antibiotic, such as rifampicin, be used to determine the half life of an mRNA?

It will stop transcription. Probes can be used to assay the concentration of a specific mRNA sequence at different points in time. The change in the concentration will be used to determine the half life of the mRNA

Can any of the eukaryotic polymerases bind to a promoter themselves?

No

Do all transcription factors bind DNA?

No. Not all have to interact directly with the DNA

What is the kinase that allows Pol II to enter the elongation phase completely?

P-TEFb (CycT1 and CDK9)

What does alpha-amanitin work?

Produced by fungus amanita phalloides (death cap mushroom). It is a potent inhibitor of RNA pol II and weak inhibitor of RNA pol III

Which eukaryotic (eRNAP) RNA polymerase is responsible for synthesizing mRNA, microRNA, and noncoding RNAs?

RNA Pol II

Which RNAP synthesizes tRNA, 5S rRNA, and 7SL RNA?

RNA pol III

What is the model of eukaryotic transcription termination?

Rat1/hXrn2 (Pac-Man looking thing) protein beginds the degradation of the RNA that is produced after the cleavage of the mRNA by the polyadenylation complex. This protein will continue to degrade the growing RNA until it reaches RNA pol II and it modifies it allosterically, causing RNA pol II to release the DNA strand.

Besides elongation, what else happens upon the phosphorylation of serine-2 of the CTD?

Splicing factors are recruited

What are the promoter elements of RNAP II?

TATA Box, BRE, Inr, DPE

What are the five general transcription factors (GTFs)?

TFIID, TFIIB, TFIIE, TFIIF, TFIIH

Besides promoter escape, what else happens upon the phosphorylation of serine-5 of the CTD?

The 5' Capping factors are recruited (5' capping comes when serine-5 is phosphorylated)

What is the CAAT and GC box?

The CAAT box binds other initiation factors. The GC box is bound by SP1 factor

Where do the first initation factors bind?

The TATA box

What level of transcription is typically the most important for regulation?

The initiation of transcription is the most heavily regulated.

What is the role of poly-A tail in eukaryotic transcripts?

mRNA stability and translational enhancement.

What unique feature is found on the subunit IIa of RNA pol II?

the CTD

How many subunits does RNA pol II have?

two (IIa and IIb) large and 8 small subunits

What is TFIIE?

two polypeptides needed for the assembly of TFIIH to form the transcription preinitiation complex