Transcription I - III Flashcards
what protein identifies transcription units?
RNA polymerases
what is the difference in transcription coupling between bacteria and eukaryotes?
in bacteria, transcription is coupled to translation. in eukaryotes, it is coupled to RNA processing
what is a transcription bubble?
DNA opening where RNA is being transcribed - consists of 10-14 unpaired bases with an RNA:DNA hybrid of 8-9 bases
what are the components of the ternary transcription elongation complex?
consists of RNA transcript, +1 coding strand, RNA polymerase, promoter and terminator
in what direction is the RNA chain elongated?
5’ to 3’
what directs RNA polymerase to the promoter in bacteria?
sigma factor, which binds to core RNA pol to comprise the RNA pol holoenzyme
what is the sigma cycle?
there are many different sigma factors which recognize different promoters, the cycle is the process whereby an RNA pol binds to a sigma, does a round of IET, then dissociates and is free to bind to another sigma
what does the sigma factor do? (3)
sigma destablizes nonspecific binding to non-promoter DNA
sigma stabilizes specific binding to promoter DNA
this accelerates the search for promoter DNA
*what are some structural changes which occur in both RNA and RNA pol II during initiation?
RNA - A base gets flipped out,
what are the basal elements of an RNA pol promoter
Upstream TFIIB recognition element (like a TATA box), initiator, downstream promoter element
TFIID is composed of what two components?
TBP and TBP-associated factors (TAFs)
How does TFIIH work?
TFIIH rotates DNA on the front end of the preinitiation complex, leading to melting and open complex formation
Mutations in what PIC protein lead to altered start site selection?
TFIIB
What does the CTD do?
CTD, or the Carboxyl terminal domain. Other proteins often bind the C-terminal domain of RNA polymerase in order to activate polymerase activity. It is the protein domain that is involved in the initiation of transcription, the capping of the RNA transcript, and attachment to the spliceosome for RNA splicing.
what’s the difference between proximal promoter pausing and abortive transcription?
proximal promoter pausing can still yield a productive transcript, but abortive cannot
why is proximal promoter pausing important?
- checkpoint to make sure 5’ cap is present
- Point of regulation: common at developmental genes and of genes involved in stimulus- controlled pathways such as heat shock protein 70
what are three broad things that general transcription factors (GTFs) can do?
recognize promoter, unwind promoter, and select the transcription start site
T/F. RNA polymerase can only proceed in the 5’ to 3’ direction
False, you get backsliding sometimes
T/F. Elongation rates can vary 10-fold for a single transcript
True, from 10-100 nt/sec, due to pausing
what are the two classes of pause sites?
Class I: rho-related, whereby RNA structures form in the exit channel and lead to clamp loosening and 3’ misalignment
Class II: weak hybrids causing pol to backtrack, shifting the hybrid to a more stable upstream register and threading the 3’ end of the transcript into the NTP entry funnel
simple termination steps in E. coli
- Transcription of GC-rich dyad sequence
- Formation of hairpin causes pausing
- Release of RNA due to instability of rU:dA hybrid
what two overarching features direct transcription termination?
- The sequence and structure of the nascent transcript
2. The activity of protein factors that interact both with the RNA and polymerase
What are 4 RNA pol II transcription regulators?
mediator, ATP-dependent nucleosome remodelers, histone modifiers and negative cofactors
what is the most abundant class of gene-specific transcription factors?
zinc fingers
what is the structure of zinc finger proteins
- multiple copies of a small beta-beta-alpha domain stabilized by a bound zinc atom
what is the structure of zinc finger proteins
Multiple copies of a small beta-beta-alpha domain stabilized by a bound zinc atom, often modular
how many DNA bp can a zinc finger recognize?
Each module in the protein functions independently and recognizes ~ 3 – 4 DNA base pairs
how many bp does a DNA sequence have to be to be considered unique in the human genome?
18
nuclear hormone receptor DNA binding domains - actions and example
Ligand activated proteins, when bound to specific seqs (hormone response elements)of DNA serve as transcription on/off switches. Examples: estrogen receptor, glucocorticoid receptor
what are three parts of a nuclear receptor?
variable region, DNA binding domain, and ligand binding domain
how does galactose induced regulation work?
Gal4 is a transcriptional activator, which sits at the cis-acting upstream activator sequence (UAS) which tethers it to the promoter. Without galactose, Gal80 binds to Gal4 in a way that prevents Gal4 from recruiting and activating RNA pol. But when galactose is present, Gal80 changes conformation and binds to a diff part of Gal4, de-repressing Gal4 and allowing it to recruit and activate RNA pol II.
what is a VP16 activation domain and how would you use it
it’s a herpes virus protein that binds human TF Oct1 - its acidic C-terminal can be transplanted to other DNA-binding domains to act as a strong activator
what is the mediator complex and what does it do?
mediator complex is part of the pol II complex, interacts with unphosphorylated CTD, recruits GTFs and Pol II, interacts with activation domains through a “fuzzy interface”
what are three characteristics of gene-specific transcription factors?
- Modular, have DNA binding domain and activation (or repression) domain
- Interact with large family of co-regulators
- Often act as dimers or higher multimers to increase binding specificity
what are enhancers?
clusters of transcription factor binding sites
what are three unique properties of enhancers?
- They can enhance transcription initiation from a distance of up to a megabase from the promoter
- They can function upstream or downstream of transcription start site
- They can enhance transcription if the enhancer orientation is inverted
what protein holds together loops which bring enhancers and promoters in close proximity
cohesin
ChIA-PET
form of chromosome conformation capture, where you do an immunoprecipitation before doing Hi-C ligation, this allows you to enrich for DNA binding protein sites in your genome
how were the estimated 40K enhancers in the human genome identified?
- Map TF binding sites and compare to TSS sites
- ID nuclease sensitive sites since TFs bind in nucleosome free regions
- Look for characteristic enhancer histone signature H3K4me1 and H3K27ac
- Clone seqs that enhance transcription
what is another method to ID enhancers which relies on assaying their functionality?
STARR-seq - chop up source genome and recombine fragments into a reporter vector , clone, transfect, then do RNA-seq to see which random pieces of your DNA act as enhancers. It’s basically an agnostic enhancer screen
what is a super-enhancer?
region of mammalian genome comprising multiple enhancers that is collectively bound by an array of TF proteins to drive transcription of genes involved in cell identity, characterized by high levels of mediator
what is a technique to image chromatin in vivo?
ChromEMT - chromatin electron microscropy tomography, where DNA is labeled with a fluorescent dye that when activated catalyzes deposition of diaminobenzidine polymers, binding OsO4 and creating contrast
what is the basic structure of a nucleosome?
wraps 147bp DNA, octet core, histones pair in special ways and have long tails that are frequently modified
T/F. Nucleosomes are mostly fixed structures.
False, they are dynamic and can transiently unwrap and rewind
what does genomic assembly of chromatin require?
chromatin remodeling complexes and ATP
what are Snf/Swi genes
chromatin remodeling complexes, needed to de-repress repressed genes
what three groups of proteins regulate the histone code
chromatin readers, writers and erasers
how does acetylation affect individual nucleosomes?
it destabilizes them by neutralizing the positive charge of lysine (lysine rich tails bind tightly to DNA and repress transcription by blocking access of factors)
what type of protein is Sir2, and what is its role?
it is a histone deacetylase, and it binds cooperatively to deacetylated chromatin to tightly package chromatin, and spread heterochromatin
what is the PRC2?
polycomb repressive complex 2, it makes multiple contacts with chromatin and is an essential spreader of certain histone marks involved in silencing.
pioneer factors?
TFs that directly bind condensed chromatin, can have both positive and negative effects on transcription, can bind their targets within nucleosomes and recruit other TFs and histone mod enzymes as well as control DNA methylation
