Lecture 15 (RR2): eukaryotic transcription Flashcards
What is the most overwhelming contributor to gene expression?
Rates of transcription –> this is why transcriptional control is so so so important.
Overview of the transcription process
1) DNA double helix locally denatures and one strand acts as a template.
2) Transcription is catalyzed by RNA polymerase. RNA polymerase will use a DNA template (moves down the template strand, starts at its 3’ to 5’) and sequentially joins the rNTPs from 5’ to 3’ on the growing strand.
3) Incoming ribonucleoside triphosphates (rNTPs) base-pair with bases in the template DNA strand.
4) Polymerization is energetically favoured because the high-energy bond between the α and β phosphates is replaced by a lower-energy phosphodiester bond.
Conventions of RNA transcription
- Speed?
- Coding sequence?
- RNA is made of? DNA is made of?
- Where does transcription begin?
- Downstream? Upstream?
- Template strand
- Speed: RNA Polymerase II advances at a rate of about 1000-3000nt/min. The longest human gene DMD (Dystrophin) takes 224 hours to transcribe.
- Coding sequence: looks exactly like the mRNA sequence except in DNA form. RNAs have U instead of T.
- RNAs are made up of ribonucleotides. DNA is made up of deoxyribonucleotides.
- The site where transcription begins is very often shown as a right angle arrow. The arrow designates that direction that polymerase is going to transcribe. (to the right in this case). We designate the transcriptional start site as +1, the first nucleotide incorporated into a growing RNA molecule.
- Upstream of the transcriptional start site, we can refer to these regions as the promoter region. The promoter regions have a lot of regulatory information that helps in telling the polymerase where it has to start its catalysis making the RNAs (information to set down the right gene at the right position). The promoter can be proximal, can be distal (regulatory info can be very far away, sometimes it can even be downstream and in introns)
- Downstream as being into the gene body from the +1 site.
- Template strand: strand that is actually read by RNA polymerase (3’→ 5’).
Three stages of transcription
- Initiation
– Polymerase binds to the promoter sequence, locally denatures the DNA, and catalyzes the first phosphodiester linkage.
– Rate limiting step in transcription process (setting up the environment so that the appropriate proteins and RNA polymerase will access the gene sound in the promoter region). - Elongation
– In this phase, RNA polymerase is processive. It continues transcribing through the body.
– Polymerase advances 3’->5’ down the template strand, denaturing the DNA and polymerizing the RNA.
– In dystrophin, it can fall off prematurely, but it’s gotta get to the end. - Termination
– Polymerase recognizes a stop site, releases the completed RNA and dissociates from DNA.
Differences between eukaryotic and prokaryotic transcription.
1) Eukaryotic DNA is packaged into chromatin, which in the condensed state (default state) results in a promoter that is inaccessible to RNA polymerase. Transcription can only occur when DNA is decondensed and dissociated from the histones around which it is wound.
2) Transcription and translation cannot occur at the same time because eukaryotes have a separate compartment for the DNA (nucleus).
3) Prokaryotic cells: genes with common function are arranged linearly and are transcribed together on a single mRNA (polycistronic). This means that they have a number of gene products that (which each encode different proteins) are located linearly next to each other, between one promoter and terminator (in the same region of DNA) - even though they all give rise to different proteins. These are called operons. Considered polycistronic - can use multiple transcription units to make multiple gene products from the same RNA precursor.
a) Eukaryotic cells: genes with common functions can be scattered throughout the genome, on different chromosomes and can therefore be transcribed and regulated separately from one another.
b) Eukaryotic mRNA is always monocistronic: one mRNA = one protein.
4) Eukaryotes have three types of RNA polymerase:
RNAP I: transcribes rRNA
RNAP II: transcribes mRNA
RNAP III: transcribes tRNA
5) Messenger RNA requires extensive modification before being translated into protein (maturing of RNA).
6) Promoters are more complex but also have a consensus sequence similar to that in prokaryotes. This is called the TATA box and is located about -30 nucleotides upstream of the transcriptional start site.
7) RNA polymerase needs the help of transcription factors to bind to DNA and start transcribing. Instead of a simple sigma factor, several transcription factors (TFs) bind to the promoter to recruit RNA Polymerase II. DNA binding proteins regulate the rate of RNA synthesis by enhancing or impeting the ability of RNA polymerase to bing to the promoter region.
8) Elongation is similar to prokaryotes but termination can be different (depends on RNA Polymerase type).
The three different RNA Polymerases and their function.
RNA polymerase 1:
- Completely insensitive to mushroom –> Amanitin Phalloides –> mushroom toxin α-amanitin.
- Responsible for forming most of the RNA in the cell, the ribosomal RNA (rRNA). Transcribes genes encoding pre-rRMA, which is processed into 28S, 5.8S and 18S rRNAs.
- Located in nucleolus
RNA polymerase 2:
- Sensitive to mushroom
- Give rise to protein coding genes, the mRNAs.
- Responsible for the generation of small nuclear RNA that are critical for RNA splicing.
- Also make small interfering RNAs
RNA polymerase 3:
- Responsible for the transcription of tRNA
- Also makes one rRNA called 5S rRNA,
- Makes an array if small stable RNAs, one single SN RNA called u6 (involved in RNA splicing) and a bunch of other small RNAs (including signal recognition particle - SRP).
All three eukaryotic RNA polymerases share common
features:
-They all exist in multimeric complexes (two large subunits + smaller subunits)
-Some show significant similarity to bacterial subunits
-Most are essential…
**The CTD of RNA Pol II is phosphorylated during transcription in vivo.
Prokaryotic vs Eukaryotic RNA Polymerase
Bacterial RNA Polymerases:
* Bacterial RNA polymerases are composed of two related large subunits (β’ and β), two copies of a smaller subunit (α), and one copy of a fifth subunit (ω). The last subunit is not essential for transcription.
Eukaryotic RNA Polymerases:
* Have the same basic structure as mentioned in the Bacterial RNA polymerase section but also a bunch of other smaller subunits critical for function.
* They all work the same way: they take up a DNA template and they will spit out RNA.
* The clamp is an important structure in eukaryotic polymerase. It is on the large subunit RBP 1. It is important because the enzyme interacts with the DNA and then locally melts it and starts to generate the RNA molecule (the RNA-DNA hybrid between RNA and the template strand). When this forms it changes the conformation of the clamp such that it shuts over the DNA and holds the RNA polymerase to that DNA molecule.
* Another protein that helps keep the clamp closed is called GSIF which also plays a role in elongation.
What is the largest subunit in RNA Pol II
- The largest subunit in RNA Pol II is an essential Carboxy-Terminal Repeat (CTD).
- RNA polymerase II contains a unique domain at the carboxyl end of its RPB1 subunit (large subunit). The domain is made of seven amino acids repeated many times: Tyr-Ser-Pro-Thr-Ser-Pro-Ser
- Yeast RNA Pol II has 26 of these repears and needs at least 10 to survive.
Analysis of polytene chromosomes from Drosophila salivary glands:
* a time of active transcription, indicates that the CTD is also phosphorylated during in vivo transcription.
* The large chromosomal “puffs” are regions where the genome is very actively transcribed. Where transcription is occuring.
* Staining with antibodies specific for either the phosphorylated or nonphosphorylated CTD:
- Phosphorylated CTD is stained red; unphosphorylated CTD is stained green. (overlap is yellow).
- Where the gene is stained red, it means that RNA polymerase II might be associated with those genes, but it’s not actively transcribing.
The clamp
- Conformational change that takes place during elongation on RNA pol II to hold the polymerase to the transcript.
- Clamp makes it so that the polymerase WILL NOT leave the transcript. Unless something tells it to leave. Generally, it will stop and try to repair before it actually leaves.
- it is on RBP1
- GSIF helps with clamp
more info
All RNA polymerase II have a carboxy terminal end not domain.
RNA Pol II is not fully engaged in elongation until NELF is removed.
RNA pol II has to be brought down to the right places in prokaryotic and eukaryotic transcription → it needs to be directed (how does it get to the correct place?).
Specific genes need to be transcribed at the right time.
