Week 5 Part 2: Transcription 2 (Eukaryotes) Flashcards
Though both eukaryotes and prokaryotes both have mRNAs, rRNAs, and tRNAs, what makes the two different?
Eukaryotes have more of these RNAs within their cells
How can you tell that the RNAP being referred to exists within a eukaryote?
If it has a number beside its name (i.e. RNA polymerase I)
What are the three RNAPs in eukaryotic cells and what genes to they transcribe?
- RNA polymerase I: most rRNA genes
- RNA polymerase II: all protein-coding genes, miRNA genes, plus genes for other noncoding RNAs (e.g., those of the splicesosome)
- RNA polymerase III: tRNA genes, 5S rRNA gene, genes for many other small RNAs
RNAP II can easily be seen as the eukaytoes version of bacterial RNAP, what is one way they differ?
Bacterial RNAP has 5 subunits, eukaryotic RNAP II has 12
What does RNAP II have that cannot be found in bacterial or other eukaryotic RNAPs?
a special carboxyl terminal domain (CTD)
What are transcription factors?
Transcription factors is the eukaryotic RNAP version of bacterial RNAP’s sigma factor/subunit (i.e. it helps initiate transcription)
When looking at the structure of DNA, what do eukaryotic RNAPs have to deal with that bacterials RNAPs do not?
Chromosomal structures
What is the TATA box?
The TATA box helps position RNAP II and is usually found ~30 bp upstream from start site for transcription
What does it mean when it is said eukaryotic promoters are more variables than bacterial?
They will have 1 or more specific sequences called elements which bind to specific locations. Elements are recognized by specific general transcription factors which help position RNAP
What is the first step in the intitiation of transcription?
The binding of TBP (TATA binding protein) subunit of TFIID (transcription factor for RNAP II) to TATA box sequence. This mobilizes the binding of TFIIB complex adjacent to TATA box. This bends DNA.
What happens after other transcription factors bind after the bending of DNA?
RNAP II together with other TFs, will be able to bind in correct orientation at transcription start site. There is also helicase activity & phosphorylation of C-Terminal Domain (CTD) of RNAP II – both of which are performed by TFIIH (the H indicates helicase)
What is an enhancer? What is its role in transcription initiation?
The enhancer is the binding site for the activator protein. It helps activate transcription.
Overall, describe the steps in the initiation of transcription
- TBP a subunit of TFIID binds to the TATA box promoter in the minor groove, bending and distorting the DNA
- This attracts the other transcription factors, which help to orient and bind RNAP II to the DNA
- The helicase activity of TFIIH uses ATP to pry apart DNA strands at the transcription start site
- TFIIH also phosphorylates the C-terminal domain of RNAP II, activating it so that transcription can begin
What does the CTD of the largest subunit in RNAP II consist of?
Tandem repeats of 7 amino acids. These amino acids are: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A human enzyme has 52 repeats.
When RNA polymerase II is activated via phosphorylation, what is phophorylated?
Adding phosphate groups on S (Ser) located on the CTD
How many proteins are invoved in initiating eukaryotic transcription?
> 100 subunits of many protiens
What are the three main steps of mRNA processing?
- Addition of 5’ cap
- Removal of introns (splicing)
- Processing and polydenylation of 3’ tail
Phosphorylation of C-terminal tail of RNA Polymerase II results in binding of:
- RNA processing proteins
- Additional phosphorylation of CTD, including Ser 2
What is 5’ pre-mRNA capping?
- Helps protect RNA from exonucleases
- Completed before mRNA fully transcribed
What are UTRs?
Untranslated regions
How are introns removed from pre-mRNA?
This is done by the spliceosome. It is a two step process:
1. Branch-point A attacks the 5’ splice site
2. 3’ of one exon reacts with 5’ of next exon to release intron
Describe the RNA dependent catalytic mechanism that allows for intron splicing
- The 2’ OH group of the ribose sugar is not present in deoxyribose
- This group is necessary for the formation of the lariat structure in intron splicing
The splicing reaction for most eukaryotic pre-mRNAs requires….?
snRNPS
What do spliceosomes contain?
snRNAs bound to protein – snRNPs, plus other associated proteins
What is added when splicing is complete?
the exon junction complex is added (these are proteins that sit where introns used to be)
True or False: There are special sequences required for accurate mRNA splicing
True, and within these sequences lies the A that forms the lariat
What nucleotide sequences does Y, R, and N stand for?
T: C/U
R: G/A
N: A/U/C/G
What does alternative RNA splicing increase?
It increases the number of gene products, this means that splcing differently produces different mRNA
Describe examples of abnormal splicing
- A single-nucleotide change that destroys a normal splice site, thereby causing exon skipping (e.g. mRNA with an exon missing)
- A single-nucleotide change that destroys a normal splice site, thereby activating a cryptic splice site (e.g. a mutation causes a splice to end more upstream than required causing an extended exon)
- A single-nucleotide change that creates a new splice site, thereby causing a new exon to be incorporated (e.g. extra exon in between introns)
What does 3’ end processing consist of?
- Consesnsus sequences direct cleavge and polydenylation of the 3’ end
- 3’ end processing proteins move from CTD to mRNA
- Cleavage and addition of a poly-A 3’ tail along with Poly A-binding proteins result in the mature mRNA
After mRNA matures (5’ cap and 3’ poly-A tail added), where does it go? And what does this mean for protein synthesis?
It is exported from the nucleus to the cytosol. This means that mRNA is translated in the cytoplasm, where protein synthesis takes place.
