Lecture 8 - Features Of eukaroyotes Flashcards
Start point
The position on DNA corresponding to first base incorporated into RNA
RNA polymerase I
Location: Nucleolus RNA products: pre-rRNA —> 5.8S, 18S and 28S rRNA
RNA polymerase II
Location: nucleoplasm RNA products: pre-MRNA and some snRNAs
RNA polymerase III
Location: nucleoplasm RNA products: tRNA, 5S rRNA, U6 snRNA (spliceosome) and 7SL RNA (signal recognition particle)
What subunits are present in RNA polymerase I,II,III?
Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12
How many subunits in Archaeal?
13 subunits
How many subunits in E.coli?
5 subunits
TFIIA
Subunit: 2 Stabilises TBP and TFIID binding Blocks the inhibitory effects of TAF1 and other proteins
TFIIB
Subunit:1 Stabilises TFIID- promoter binding. Contributes to transcription start site selection Help recruit RNA polymerase II TFIIF to the core promoter
TFIID
Subunit: 1 and 14 Bind TATA element and deforms the promoter DNA. Platform for the assembly of TFIIB and TAFd. Binds Inr, MTE, DPE and DCE promoter elements
TFIIE
Subunit: 2 Helps to recruit TFIIH to the core promoter and is required for promoter melting
TFIIF
Subunit: 3 Bind RNA polymerase II and is involved in recruiting the polymerase to the pre-initiation complex. Required to recruit EFIIE and EFIIH to the pre-imitation complex
TFIIH
Subunit: 10 Functions in Transcription and DNA repair. It has kinase and helicase activities and is essential for open complex formation
RNA Polymerisation
The 3’OH group of the last ribonucleotide asses to the chain reacts with an incoming ribonucleoside 5’triphosphate
How many RNA polymerase a do eukaryotes have?
3
Each RNA polymerase in eukaryotes is specifically dedicated to ?
rRNA,mRNA or tRNA
Eukaryotic RNA polymerase requires
A number of transcription factors Factors to modify chromatin structure to allow RNA polymerase access
RNA polymerase I no of subunits
14
RNA polymerase II no of subunits
12
RNA Polymerase III number of subunits
17
Do eukaryotic RNA Polymerase share subunits?
Some but not others
Shared subunits between eukaryotic RNA Polymerase I,II,III
Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12
Are there RNA polymerase II homologous in RNA polymerase I and III?
Many RNA polymerase II subunits have homologs in RNA polymerase I and/or III
Bacterial RNA polymerase subunits has at least … homolog in each eukaryotic polymerase
One
RNA polymerase separates the two strands of DNA in a transient …
Bubble
Role of the transient DNA bubble?
It uses one strand as a template to direct synthesis of a complementary sequence of RNA
What is the length of transient DNA bubble?
12 to 14bp
What is the length of RNA-DNA hybrid within transient DNA bubble?
8 to 9bp
What are the 3 stages of transcription ?
Initiation, elongation and termination
What is the role of RNA polymerase in transcription?
RNA polymerase binds to a promoter site on DNA to form a closed complex RNA polymerase initiates transcription after opening the DNA duplex to form transcription bubble (open complex) RNA polymerase then escapes the promoter During elongation the transcription bubble moves along DNA The RNA chain is extended in 5’ to 3’ direction by adding nucleotides to the 3’end When transcription terminates: The DNA duplex reform.
RNA polymerase dissociates at a terminator site
What are the two forms of transcription initiation ?
Focused and dispersed
RNA polymerase cannot initiate transcription from specific start site without the assurance of other proteins such as?
Transcription factors
What do each RNA polymerase have ?
Own specific set of transcription factors
What is the role of transcriptional factors that are unique to each RNA polymerase?
Factors to assist in locating Pol I, II, III transcriptional start site
Approx percentage of vertebrate core promoters that are dispersed promoters?
70%
Order of assembly of pre-initiation complex?
TATA. TFIID TFIIA
TFIIB TFIIF pol II TFIIE.
TFIIH
Structure of TATA binding protein (TBP)
Saddle shaped and bend DNA by 80 degrees. This is the subunit of TFIID that binds to TATA box
What does the interaction between TBP and TATA box Involve?
Conformational change in both TBP and the TATA box
Where do TBP and TATA interact ?
The minor groove
Do TBP-associates factors (TAFs) bind promoter elements other than TATA?
Yes
TBP associated factors (TAFs)
Additional proteins present in TFIID are called TAFs TFIID Contain a core set of 13 TAFs.
TAFs are required for high level of transcription and to transcribe genes that lack a TATA box
What happens in TATA Less promoters?
It is the TAFs that bind to regulatory elements
Where does TBP bend the TATA box?
C terminal domain of TFIIB
What happens in the N terminal domain of TFIIB?
Brings the complex to RNA polymerase II and positrons the transcription initiation site in the active site of RNA polymerase
What does the RNA polymerase II-TFIIB-promoter complex recruit ?
TFIIE which then recruits TFIIH
What does the helicase activity of TFIIH unwind?
DNA in the vicinity of initiation site
What does the TFIIF capture?
non-template strand after unwinding
Where does the template strand descend to?
Active site
What is open complex formation?
Initiation of transcriptional bubble
What is RNA chain initiation?
First 2 ribonucleotide line up on template strand in the transcription bubble and RNA polymerase catalysed phosphodiester bond formation
What is abortive transcription?
Formation of short transcripts which are released
What is promoter escape?
Chain extensions beyond 7 ribonucleotides trigger TFIIB release and formation of transcriptional elongation complex
RNA polymerase must be … for elongation?
Phosphorylated
What is the important role of carboxyl terminal domain (CTD)?
Transition from initiation complex to elongation complex
How many subunits do TFIIH have?
10
What is the structure of TFIIH?
10’subunits, ring shaped 5’—>3’ and 3’ —> 5’ helicase activities Cyclin dependent protein kinase activation
What does ser-5 phosphorylation of RNA pol II permit ?
Promoter clearance
What are phosphorylated in RNA polymerase II during elongation?
Ser-2 and Ser-7
What is more stable than initiation complex?
Elongation complex
How many base pairs are melted to form the transcription bubble ?
14bp
How many nucleotides within bubble are paired with RNA chain?
First 8 nucleotides
Where does the transcription bubble extend from?
-12 to +2
What is phosphorylation of CTD involved in?
Processing MRNA transcript
What does phosphorylation of CTD provide ?
Binding/recognition sites for mRNA processing
Does RNA polymerase have a steady pace ?
No
What happens to chain elongation at pause sites?
Temporarily delayed
What May pausing of chain elongation cause?
Pausing may lead to arrest and termination arrests is an important step in proof reading
What re-activates arrested RNA polymerase II?
TFIIS
What form of promoter is RNA polymerase I?
Bipartile promoter
What does RNA polymerase I consist of?
Upstream promoter element (UPE) Core binding factor SL1
What does the factor UBF1 do?
Wrap DNA around a protein structure to bring the core and UPE into proximity
What does SL1 Include?
TATA binding protein that is involved in the initiation by all three polymerases
RNA polymerase I binds to what at core promoter?
UBF1-SL1
What does RNA polymerase III use?
Downstream and upstream promoters
How many types of promoters does RNA polymerase III have?
3
How many internal promoters does RNA polymerase III?
2
What are internal promoters?
Have short consensus sequences located within transcription unit and cause Initiation to occur at a fixed distance upstream
What are upstream promoters?
Contain 3 short consensus sequences upstream of the start point that are bound by transcription factors TFIIIA and TFIIIC bind to the consensus sequences and enables TFIIIB to bind at start point