Week 4: Transcription Flashcards
Describe a genetic transcription locus including the startpoint, template, non-template, coding and non-coding strands.
The startpoint of any gene is delineated by a +1 point on the gene, and is where transcription actually begins. Upstream of the startpoint is the promoter region, which is usually within a few hundred BPs of the startpoint. The promoter is a NON-palindromic region of DNA that is orentation-specific.
The template strand of DNA is the strand read 3’ to 5’ by RNA polII. This is also called the non-coding strand, because it is the complement of the RNA that will be formed (i.e. the G on a DNA strand will form a C on the RNA strand, and T residues are swapped for U residues on RNA)
The non-template strand is the strand that is NOT read by DNA polII. This is called the coding strand because it is identical to the RNA transcript (with the exception of T/U swapping)
What is the sequence of recruitment and initiation for pol II and how do you remember this?
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TFIID binds to the TATA box
TFIIB and A bind to TFIID
TFIIF and Pol II bind to the complex of established TFII proteins
TFIIE and TFIIH bind, and helicase activity in TFIIH unwinds DNA at the promoter
Kinase activity in TFIIH phosphorylates the polymerase at the CTD of Pol II enabling it to transcribe
Define a promoter, enhancer, and silencer, and describe the DNA elements likely to be found in each, as well as the roles they play in transcription of Pol II-dependent genes
Promoters, including the TATA box, define where DNA transcription should begin. They are usually located about 30 bp upstream of the initiator sequence. Between the two is the untranslated region.
Enhancers can be located thousands of bp upstream of the start site. They can form DNA loops that can construct remodeling and modification complexes that allow them to act on the TBPs at the promoter region, helping initiate transcription
Gene silencing can happen through several mechanisms, including HATs and HDAs, which affect histone acetylation. Histones can also be ejected entirely or in dimers, or can be replaced with different versions.
What are the basal trancription factors and how do they function?
Basal transcription factors are necessary for eukaryotic TFs. They are usually a part of the pre-initiation complex with RNA pol. They are most critical in helping RNA pol bind to DNA, as RNA pols often have weak DNA binding capacities and rely on basal transcription factors to help recruit them. TATA binding protein (TBP) is a basal transcription factor useable by all 3 types of RNA polymerase, if the gene being transcribed has a TATA box.
Explain the activities of chromatin remodeling complexes, HATs and HDACs
Chromatin remodeling complexes use acetylation/deacetylation to help nucleosomes slide down a DNA strand to allow for selective transcription of a site, ejection of an entire nucleosome or H2A/B dimer, or replacement of the H2A/B dimer
Histone acetyltransferases (HATs) add acetyl groups to histones to allow for uncoiling and transcription
Histone deacetylases (HDACs) remove acetyl groups, allowing DNA to re-associate with histones/nucleosomes
What are the two types of hormone receptors?
Monomeric type I receptor (sex hormones and glucocorticoids) - NR-Hsp70s found in the cytoplasm complex with hormone signals that cross the cell membrane. This causes Hsp70 to dissociate, the NR receptor dimerizes and moves into the nucleus to expose a region for transcription.
Type II (thyroid hormone receptor; TR) receptors are found in the nucleus bound to corepressor retinoid x receptors (RXR). When the hormone binds, the corepressor dissociates and transcription can begin.
How does DNA methylation affect transcription?
DNMTs transfer methyl groups to cytosine in order to inhibit DNA transcription. Methylation prevents the binding of both transcription factors and Pol II.
In cancer cells, tumor suppressor genes are often incorrectly methylated/repressed so that they cannot be expressed properly, allowing tumors to proliferate.
What are the 5 major steps of transcription?
(1) Pol II is recruited to DNA by transcription factors
(2) The transcription bubble is formed by TFIIH and TFIIE. TFIIH is also responsible for CTD phosphorylation
(3) The CTD is phosphorylated during initiation. The polymerase escapes the promoter and begins elongation.
(4) Transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate
(5) Elongation factors dissociate. The CTD is dephosphorylated as transcription terminates, facilitated by termination factors like palindromic sequences/DNA hairpins
What ions are present in the active site of RNA Pol II?
Magnesium ions (Mg2+), which help position the incoming nucleotide by transiently binding the alpha/beta phosphate groups on the incoming NTP
What do RNA Pol I, Pol II and Pol III synthesize, respectively? What do they all need to function?
RNA Pol I synthesizes pre-ribosomal RNA (pre-28S, 18S, and 5.8 rRNAs)
RNA Pol II synthesizes mRNA and miRNAs (very fast, inhibited by mushroom toxin alpha-amanitin, and can recognize thousands of promoters)
RNA Pol III synthesizes tRNAs and some small RNAs
They all need a DNA template, Mg2+ cofactor, and ribonucleotides to function
What does the promoter, RNA-coding, and termination sequence look like on DNA? What direction is the template/noncoding strand read?
The promoter is upstream of the 1+ startpoint. The RNA-coding sequence is between the startpoint and the termination site, which is a poly-A sequence that signals the end of transcription of a given gene. The DNA template strand is read 3’ to 5’.
What is a consensus sequence and what is it’s function?
A consensus sequence is a region of DNA recognized by RNA polII, such as a TATA box. It is located upstream of the initiator sequence. Another type of promoter is the downstream promoter element, which is located around +30 within the gene (DPE). Some genes have both a TATA box and a DPE, some have one and some have neither. The TATA box tends to make transcription initiation a little more specific.
Another type of core promoter is the initiator element/motif (Inr) which is a promoter located near the startpoint. These can have any sequence in the YYANWYY format, with Y = pyrimidine, N = any nucleotide, W = weak (A or T, which have only two H-bonds)
What are enhancers and where are they located?
Enhancers are DNA sequences/elements approximately 100 bp long that can be far up/downstream of the core promoter. They exist because usually the core promoters contain the minimal amount of DNA necessary to recruit the polymerase, but won’t initiate transcription quickly on their own.
When activator proteins bind to enhancer DNA, they CAN act to form a pre-initiation complex that enhances the speed of polymerase recruitment, binding and activation. The activator can loop or bend to move into close proximity to the core promoter. The activator can have a direct effect, or recruit coactivators/mediators that help RNA pol associate with the core DNA sequence.
What is mediator?
Mediator is a key polymerase activating protein that helps bridge the gap between activator proteins and the polymerase binding site.
What are silencers and where are they located? How do insulators act somewhat similarly?
Silencers are DNA elements much like enhancers, and are located far up/downstream of the core DNA start site/promoter. They act in opposition to enhancers, and slow transcription when bound by repressors (as opposed to activators)
Insulator sequences block the activity of activators
What is the mechanism for the addition of an NTP to the growing strand of RNA?
The 3’ OH of the last nucleotide on the strand will attack the alpha phosphate of the incoming NTP. THis creates a 3’-5’ phosphodiester bond, elongating RNA by one nucleotide and releasing pyrophosphate, which is instantly hydrolyzed.
What is epigenetics and how does it affect transcription?
Epigenetics affects DNA expression via the change of DNA-associated proteins and complexes, including:
Histone acetylation, deacetylation, and methylation
Chromatin (a collection of 8 histones) is also remodeled via the ATP-dependent alteration, repositioning, addition or removal of nucleosomes. They are altered only breifly so that transcription occurs in a brief window before nucleosomes are repackaged. Some examples include:
Sliding, ejection, H2A/H2B dimer ejection/replacement
What two enzymes regulate acetylation, and what types of residues to they usually act on?
Histone Acetyltransferases (HATs) add acetyl groups to basic (+ charged) amino acid side chains, preventing their interaction with negatively charged backbone phosphate groups. This forces histones to unwind for a short period of time so that transcription can proceed.
Lysine is a common target
Histone deacetylase complexes (HDACs) remove acetyl groups, allowing the histones to reassociate with DNA strands.
