1.9 - Eukaryotic Transcription Flashcards
how many RNA polymerase in bacteria/prokaryotes?
1
how many RNA polymerases in eukaryotes?
3
what class of RNA do RNA polymerase I and III transcribe? (2)
- rRNA
- tRNA genes
what class of RNA does RNA polymerase II transcribe? (2)
- mRNA
- some other non-coding RNA genes
TATA box
sequence about 25 nucleotides upstream of transcription start site
TATA box sequence
TATAAAA
role of TATA box sequence
recognition sequence for TATA-box binding protein (TBP)
where is TATA -box binding protein (TBP) located
within the transcription factor II D complex (TFIID)
what does TBP’s 2 similar structures allow it to do?
bind to DNA and cause it to bend
what does bending of DNA (due to TBP) allow for?
recruitment of additional transcription factors to help recruit RNA polymerase II to bind
what does binding of TBP and TFIID to TATA box allow?
recruitment of additional proteins to bind (TBP and TFIID = general transcription factors)
where do general transcription factors bind?
bind to additional upstream control elements which are present in the 200 base pairs upstream of transcription start site
transcription factor structure
2 domains that bind to DNA regulatory regions and to RNA polymerase through other proteins
what DNA groove to transcription factors bind to?
major groove (hence how promoter provides strand specificity)
function of 3 different types of transcription factor
same for all
role of general transcription factors
help with placement of RNA polymerase II to promoter
role of regulators (enhancers and repressors)
at long distances away from promoter can affect stability of RNA polymerase II/general transcription factor complex at promoter
how do regulators act in order to have effect on transcription?
cis-acting proteins act through mediator complexes which bind to RNA polymerase II/ general transcription factors
how much can regulators increase transcription rate?
up to 1000-fold
how can chromatin effect transcription (2)
- TATA box has to be available for protein to bind
- if DNA wrapped around nucleosome, no general transcription factors able to bind to TATA box to initiate transcription
what is required to expose promoter for transcription when it is blocked by chromatin?
chromatin remodelling
what is required for separation of DNA strands and initiation of transcription (unlike bacteria)
ATP hydrolysis
what do proteins within transcription factor II H mediate? (2)
- melting strands for RNA polymerase II
- ATP hydrolysis for separation of DNA strands and initiation of transcription
transcription factor II H structure
unlike other transcription factors, complex of proteins each with important roles in initiation of transcription
helicase
pries double helix apart and melts DNA at transcription start site
(part of TFIIH complex)
how is energy obtained for helicase prying double helix apart?
hydrolysing ATP generated by ATPase that is part of TFIIH
transcription bubble
helicase creates transcription bubble when double helix is separated to allow transcription to occur
how does RNA polymerase II escape binding to all DNA-binding proteins holding it in place at promoter? (2)
- another 2 kinases that are part of TFIIH phosphorylate the amino acids at the C-terminal of the protein
- allows RNA polymerase to disengage from general transcription factors and begin to elongate RNA transcript
(other transcription factors removed, TFIIH remains bound to continue to initiate transcription as required)
C-terminal in eukaryotes
specific to eukaryotes, not present in bacteria
why are ribosomes unable to immediately translate mRNA?
RNA transcribed in nucleus (unlike in bacteria)
what is a result of RNA having to move out of nucleus to be translated?
number of modifications required to keep RNA stable and prepare it for translation
modifications that occur for mRNA destined for translation (3)
- capping
- splicing
- polyadenylation
what does phosphorylated state of C-terminal of DNA allow for?
enzymes to bind to it in preparation for modifying RNA transcripts being transcribed
why is the 5’ end of RNA capped? (2)
- protect RNA transcripts from degradations and to enable
- nuclear export and translation
what does capping the 5’ end of RNA transcripts involve? (2)
- removal of the terminal phosphate group and addition of a guanine to form a 5’ to 5’ triphosphate bridge
- guanine then methylated at N7 position (sometimes additional methylation can occur)
when does capping of RNA transcript occur?
after 25 nucleotides have been added to the RNA transcript
when can splicing occur on pre-mRNA while translation is occurring?
as long as the 5’ end has been capped
why do introns need to be removed before translation can occur?
introns do not code for amino acids, need to be removed so only exons remain
size of introns compared to exons
introns longer than exons, sometimes longer than all exons together
(in humans exons average 150 nucleotides, introns average 1500 nucleotides)
how many exons can genes have?
one or many, number correlated with species and protein complexity
intron effect on mRNA transcription in eukaryotes compared to bacteria
takes significantly longer in eukaryotes due to presence of introns
how does splicing add diversity to gene expression?
allows for different versions of RNA transcripts and consequently proteins to be produces from same gene
(can be useful as isoforms of protein needed in different times and/or cells)
how do spliceosomes know where to splice introns?
at each end of introns in primary transcript are splice site indicating boundaries of the intron
nucleotides at 5’ and 3’ end of introns (2)
- 5’ = GU
- 3’ = AG
branch site within intron
focussed around a single A, important for excision and normally closer to 3’ end than 5’ end
role of branch site for intron removal (6)
- 2’OH group on the A that is part of branch site bends transcript
- performs nucleophilic attack on first G of the 5’ splice site, causing it to be cut
- frees 5’ end of intron, then becomes covalently linked to 2’OH of the A on branch point (lariat intermediate)
- OH group of 5’ exon then does nucleophilic attack on last G in 3’ splice site
- forms intron lariat which is removed from mRNA
- joins 2 exons together to be part of mature RNA transcript
spliceosome
complex protein and RNA structure responsible for undertaking cutting of the transcripts
spliceosome structure
small nuclear RNAs (snRNA) and over 150 different proteins
role of small nuclear RNAs (snRNA) in spliceosome
involved in helping to mediate bringing the splice and branch sites together (like an enzyme)
spinal muscular atrophy (SMA)
group of diseases caused by a loss of SMN1 that decreases motor neuron survival
how is transcription terminated?
RNA polymerase II reaches termination sequence where it stops transcribing and synthesising RNA
what does RNA polymerase II transcribe before is disassociated from DNA and RNA?
polyadenylation signal - allows for enzymes involved in these processes to bind
role of 3’polyadenylation site
(RNA cleaved to expose site)
poly-A polymerase (PAP) will bind to 3’ end of transcript and add around 200 adenines without a template (poly(A) tail)
role of addition of poly(A) tail
helps provide stability to mRNA and promote translation
why does translation require 5’cap and poly(A) tail? (2)
- 5’ cap binding protein recognises 5’ cap
- multiple poly(A) binding proteins bind along entirety of poly(A) tail
required process for RNA to be exported from nucleus (with all the proteins attached) (3)
- capped
- spliced
- plyadenylated
what do the attached proteins on RNA transcript signal?
signals to nuclear pore complex to move RNA transcript from inside nucleus to cytoplasm where it can be translated
