Genetic information - Transcription Flashcards
what are the 4 main levels at which transcriptional control is exerted
Binding of RNA polymerase: promoters and transcription factors
Long range control: locus control regions
Chromatin remodelling: histones and histone deacetylases
DNA methylation: CpG islands and imprinting
what genes does RNA polymerase I transcribe
28s RNA
18s RNA
58s RNA
what genes does RNA polymerase II transcribe
all genes that encode snoRNA
most snRNA
what genes does RNA polymerase III transcribe
5sRNA
tRNA
some snRNA
and other small RNA
All genes that are transcribed and expressed via mRNA are transcribed by which polymerase
RNA polymerase II
what are the differences between bacterial and eukaryotic RNA polymerase II
eukaryotic - 12 subunits instead of 5
cannot initiate transcription - no σ factor
has to deal with DNA packed into nucleosomes
function of transcriptional activators
Transcriptional activators help attract RNA polymerase II to the promoter, help regulate rate and tissue specificity of gene expression
what does the initiator region highly conserve
pyrimidines
where does RNA Pol bind to
TATA box
what is elongation
Release RNA Pol II from promoter
what is TFII
a general transcription factor of RNA polymerase II
how is a pre-initiation complex (PIC) formed
The first transcription factor TFIID binds the TATA box
Other transcription factors join it
Once some transcription factors have attached to the TATA box
- More transcription factors bind
how is elongation achieved
C-Terminal domain (CTD) phosphorylated
Conformation change – tightens grip
General TFs dissociate
Acquires new proteins – including elongation factors that help process the RNA and increase elongation rate
how do the transcription factors involved in the formation of PIC function to achieve this
IID contains TBP which binds to the TATA box
IIA stabilises IID binding to promoter
IIB binds initiation sequence
Pol II binds IIB
IIE stimulates transcription
IIH has kinase and helicase activity
what does a TATA less core promoter contain
contain an INR and DPE
what is a DPE
downstream promoter element
located at 28+ - 32+
has the sequence AGAC
recognised by TFII I
what causes growth factor deficiency
mutation in Pit-1 transcription factor
DNA sequence motifs bound by general transcription factors
the general TF, Sp1 binds to GGGCGG
Sp1 is found in all cell types
. DNA sequence motifs that confer tissue specific expression
MyoD is a muscle-specific transcription factor
Note all cells have CANNTG but only tissue specific cells have the MyoD TF expressed.
. DNA sequence motifs that confer response to particular stimuli
Oestrogen
what are enhancers
regulatory sequences that act at a distance
on an immunoglobulin enhancer what is the function of E1-E4
B-cell specific Transcription Factor
on an immunoglobulin enhancer what is the function of C1-C3 and Oct
C1-C3: General Transcription Factor
Oct: B cell specific Transcription Factor
what determines wether a gene is switched on
TF binding
features of a zinc finger DNA binding domain
contain a loop of 23 amino acids
usually have multiple zinc fingers
the linker between the zinc fingers is 7-8 amino acids
role of zinc in DNA binding
do not directly interact with the DNA but is essential for folding of the DNA
zinc fingers bind to both major and minor grooves
features of a helix-turn-helix (homeodomain)
homeodomains are 60 amino acids long
The C terminal alpha-helix 3 is 17 amino acid’s and lies in the major groove
Helices 1 & 2 point away from the DNA
features of basic positively charged binding domains
transcription factors with basic binding domain cannot bind to DNA alone
they must dimerise to bind to DNA
features of leucine zipper proteins
bind to major groove of DNA with hetero/homodimer via extended α-helices
contain leucine or hydrophobic amino acid in every 7th position in the C-terminal of binding domain
hydrophobic residues form coiled coil domains required for dimerisation
what are the different types of activation domains
Acidic amino acids - Gal 4
Glutamine rich - Oct 2
Proline rich - Jun
how do transcription factor activation domains work
direct interaction with the PIC
recruitment of co-activators
what co-activator do transcription factors recruit to modify histones and how does it do it
histone acetylase
how do inhibitory domains work
binds to DNA and prevents TFs with activation domains from binding
bind to PIC and block transcription with its inhibitory domain
recruitment of co-repressors
name a co-repressor and how it inhibits transcription
histone de-acetylase (HDAT)
- Removes acetyl group of histone units
- restores +ve charge of histone
Close down DNA
Shutting off transcription
what is step1 of RNA splicing
cleavage at the 5’ splice site
The ribose hydroxyl group (OH) of the A within the branch point acts as a nucleophile and attacks the 5’ splice site
what is step 2 of RNA splicing
cleavage at the 3’ splice site and joining of the exons
The newly liberated 3’OH group of the 5’ exon becomes a nuclophile and attacks the phosphoryl group at the 3’ splice site
what is RNAP II
carries many of the proteins involved in splicing
features and structure of splicesome
Comprised of both RNA (5) and protein (150)
RNA component: small nuclear RNAs (snRNAs)
U1 U2, U4, U5, U6
(100-200 nucleotides long)
Each RNA is complexed with 6-10 proteins to form
small nuclear ribonuclear proteins (snRNPs)
Different complexes come in at different stages
Uses ATP
structure and function of U1
First U1 snRNP binds to 5’ splice site
U1 snRNP consists of U1 snRNA + proteins
function of U2 and what does it create
binds to branch site - displaces BBP
aided by U2AF
Branch point A residue is excluded creating a single nucleotide bulge
which class of RNA splicing has the most eukaryotic genes
nuclear pre-mRNA splicing
structure of nuclear pre-mRNA splicing
Two transesterification reactions (branch site A)
what are the features of group II self-splicing
Some eukaryotic genes (rare)
Two transesterification reactions (branch site A)
RNA enzyme encoded by intron (ribozyme)
what do self splicing introns do
folds itself into a specific conformation within the mRNA and catalyses the chemistry of its release
features of group I self-splicing
Some eukaryotic genes (rare)
Two transesterification reactions (branch site G)
RNA enzyme encoded by intron (ribozyme
same as group II
what is important for splice site selection
SR proteins
features of SR proteins
SR proteins are rich in serine & arginine
SR proteins bind to sequences called exonic splicing enhancers (ESEs)
SR proteins recruit the splicing machinery to the nearby splice sites
what is alternative splicing
A process by which the RNA can be spliced in more than one way
This can result in the formation of 2 or more proteins from the same gene
name an example of a gene that is alternatively spliced
Calcitonin gene related peptide (CGRP)
function of cytosine deaminase
amino group on nucleotide ring is removed
converts cytosine into uracil
function of adenosine deaminase
converts adenosine into inosine
how does RNA editing in glutamate receptors effect the cell: adenosine to inosine
alters the Ca2+
permeability of the channel
what is thalassemia
genetic disorder in which there is decreased production of one of the globin chains found in haemoglobin
what is the difference between β0 and β+ thalassemia
β0 thalassemia major (both genes are affected)
- Develops severe anaemia starting at a few months of age
- Can only survive with the help of frequent blood transfusions
β+ thalassemia minor non-life threatening form of the disease
- Mild anaemia and does not generally require treatment
how does β0 thalassemia prevent globin synthesis
creation of a cryptic splice site
nonsense protein
what is the purpose of the addition of a 5’cap to mRNA transcript
protection and marking a mRNA
purpose of 3’ processing and polyadenylation to RNA transcript
marking as the end of mRNA and protection
what is the first step in the addition of a 5’ cap to RNA
addition of 7-methyl-guanosine to the 5’ end of RNA
when is RNA capped
as soon as it emerges from the exit channel of RNA Pol II
where are capping enzymes located
bound to the RNAP II’s C-terminal domain (CTD)
what does the first base at the 5’ end of RNA contain
3 phosphates:
alpha
beta
gamma
what removes the gamma phosphate
RNA triphosphatase
what is added to the 5’ end of RNA
GMP
function of guanylyltransferase
removes the beta and gamma phosphates from GTP
converted into GMP
how is guanine added to the 5’ end of RNA
it is added in reverse to the rest of the transcript
forming a 5’-5’ linkage
what does methyltransferase do in terms of 5’ capping
adds a methyl group to the guanine
why is the C-terminal domain (CTD) of RNAP II central to the process of 5’ capping
provides the binding sites for proteins involved in the post-transcriptional modification of the RNA transcript
what are the enzymes involved in co-translational capping
RNA triphosphatase
Guanylyltransferase
Methyltransferase
what is the purpose of a 5’ cap
helps distinguish mRNA from other RNA
helps mRNA be properly processed and exported from the nucleus
protects it from degradation in the cell
what is a cap-binding complex (CBC) and what is their function
they are bound to mRNA
aid in export and protect it from de-capping
serve as marker for pioneer round of translation
what is the first step in 3’ processing and polyadenylation
the 3’ end is polyadenylated by a string of adenosines ~200
function of Poly-A binding proteins (PAB)
bind to the poly-A tail and help stabilise the RNA
what are the proteins involved in cleaving the RNA
cleavage factors I/II (CFI/II)
cleavage stimulation factor (CstF)
function of exon junction complex (EJC)
binds to close to the splicing junction in association with the spliceosome
EJC is targeted by nuclear export factor I (NXF1) and nuclear export transporter I (NXT1)
RNA is transported through the nuclear pore complex
