L1 - Basal Transcription Machinery Flashcards
What are the features of nuclear RNAPI?
14 subunits
Transcribes rDNA only
What are the features of nuclear RNAPII?
12 subunits
Transcribes most nuclear genes and most snRNA and miRNA
What are the features of nuclear RNAPIII?
17 subunits
Transcribes a few short genes e.g. tRNA genes, 5S rRNA, U6 snRNA
What are are the types of non-coding genes/
rRNA
tRNA
snRNA
miRNA
How many protein encoding and non coding genes do humans have?
23000 protein
4000 non coding
What is similar about bacterial rnap and eukaryotic rnap?
Share similar cores
What can mutations in Pol3 subunits cause?
Hypomyelinating neurodegenerative disorders
How is it thought mutations in pol3 cause neurodegenerative disorders?
Maybe certain CNS cells are especially sensitive to abnormal pol III activity or pol iii products only needed in certain CNS cells
What is TBP?
TATA Binding Protein
What does TBP do?
Binds TATA sequence in promoters
Bends DNA 90 degrees
Used by all 3 pols
Provides platform for binding of other TFs
How is the pol ii preinitiation complex formed?
- TBP bends DNA to allow TFIIB to contact TFIIB recognition element (BRE) flanking TATA box
- TFIIB attracts pol ii and TFIIF
- TFIIE joins and recruits H
What are the features of TFIIH?
10 subunits (XPB, XPD,Cdk7) Dissociable kinase domain called CAK with a Cdk7 which phosphorylates CTD of pol ii
What can mutations in XPB or XPD subunits of TFIIH cause and why?
Xeroderma pigmentosum (defective repair of UV damage, extreme photosensitivity) Because TFIIH involved in DNA repair too
What does XPB helicase do?
Melts DNA and feeds template into pol ii
What does TFIIB do?
Stabilises open DNA and recognises TXN start
What is the transcription bubble and what are its features?
8-9 nts of RNA remain annealed to DNA template, as 3’ RNA extends, 5’ peels away from DNA
Melting at front of bubble balanced with reannealling at back so size constant
What are the key features of the active site of pol ii?
Template nt forms Watson crick base pairing with incoming NTP
3’ end of RNA stacks on base of incoming NTP
Mobile trigger loop forms hairpin when NTP binds
How is the phosphodiester bond formed?
NTP condensation cataltsed by two Mg2+ residues
MgA promotes deprotonation of 3’ RNA OH group
Resultant O group attacks substrate NTP phosphate
MgB stabilises transition state
Bond forms, PPi released with MgB
What speed can pol ii elongate at and with what help?
2000 per minute with elongation factors
What does TFIIS do?
allows arrested polii to resume txn when it has disengages
How does TFIIS unarrest txn?
Zinz ribbon inserts into active site to stimulate nuclease activity to cleave RNA, creating new RNA 3’ end that is correctly positioned for txn
What is RPB1 and what are its features?
Largest subunit of pol ii
Has a CTD which is of 7 residue repeats
Number of repeats varies with species, humans have 52
What does RPB1 do?
Provides docking platform for proteins according to its phos state
Why does the phosphorylation state of RPB1 matter?
Varies through txn cycle allowing dynamic recruitment of proteins
What happens during the dynamic phosphorylation cycle of pol ii?
Recruited unphos
H phos Ser5 and 7 when txn initiates
During elongation Ser5 gradual unphos and Ser2 gradual phos
What function does the dynamic phosphorylation cycle give?
CTD code shows where polii is along a gene
How is the CTD code read and used?
Mediator binds:
- unphos CTD - brings mRNA capping enzymes
- Ser5p and Ser2p recruits elongation and splicing factors
- Low Ser5p and high Ser2p recruits processing and export proteins
What do most mRNAs end in?
AAUAAA
What does CPSF do?
Recognises end of mRNA, cleaves and adds polyA tail
What does the poly A tai ldo?
Enhances export, stability and translation
What happens after poly adenylation?
Exonuclease attacks unprotected 5’ end of RNA still attached to poly ii and then DNA released from DNA
What does pol I do?
Transcribes single rDNA gene to make rRNA precursor
How is pre-rRNA processed?
28S, 18S and 5.8S rRNAs
How much of a cells RNA is rRNA?
80%
What are NORs?
Nucleolar organiser regions
200 copies of rDNA in tandem repeats clustered on five human chromosomes
What happens in cancer and how can it be diagnosed?
Synthesis of rRNA elevated and nucleoli form around it.
Increased number and morphology of nucleoli indicator of tumourigenesis
What factors contribute to the prognosis of cancer?>
Smaller nucleoli and slower growth = better prognosis
How is Pol 1 txn initiated?
UBF is TF
SL1 is tf found at promotor
Complex of pol 1 with TIF-IA recruited by SL1
What occurs in pol I elongation and termination?
Stops at termination sites boun dto TTF-1
PRTF assists release
What are the TFs for pol iii and what are their functions?
C binds promoter
C recruits B
iii recruited by B
Why does pol iii have no termination factors?
Can return to start with out dissociating
What are the similarities of the pols?
TBP is part of SL1 and TFIIIB
Several subunits found in pol1 and 3 correspond to TFs of pol ii
What is interesting about the evolution of pols 1 and 3?
Involved attachment of txn factors that interact more loosely with pol 2
How is each pol inhibited?
a-amanitin toxin
1 is resistant
2 is inhibited by low doses
3 is inhibited by intermediate doses
What controls are used when using toxin to inhibit pols?
GAPDH as + control to confirm treatment efficacy
tRNAtyr as - as pol III is les sensitive to toxin than 2
