Transcription Flashcards
What is the difference between DNA and RNA?
- DNA contains deoxyribose, RNA contains ribose
- in RNA, U (intead of T in DNA) base pairs with A
- DNA double stranded, RNA single stranded (in eukaryotes and prokaryotes)
What is RNA polymerase (RNAP)
multi-subunit complex that synthesizes RNA using a DNA template => transcription
- Catalyse the formation of phosphodiester bonds between ribonucleotides
- Moves stepwise along DNA, unwinding helix
overview of how RNAP perform using DNA as template?
- RNAP unwinds DNA with its active site
- one of the two DNA strands acts as a template for complimentary base pairing of RNA monomers (ribonucleotides)
- RNAP catalyses the formation of phosphodiester bonds between ribonucleotides
- RNA then dissociates from DNA, DNA double helix reforms
- single stranded RNA released
What provides the energy for RNA polymerisation?
The energy stored in the P-P bonds (phosphoanhydride bonds) of ribonucleoside triphosphates (ATP, GTP, CTP, UTP)
What are the base parings in RNA?
- Adenosine pairs uracil
- guanine pairs cytosine
in what direction is RNA synthesised?
5’ to 3’
RNA does not always enter translation (process where mRNA is translated into proteins), what is the function of those RNA?
because RNA is single stranded, they can be folded into precise structures:
- can have structural and catalytic functions like proteins
- e.g. rRNA: forms basic structure of ribosomes and catalyse protein synthesis
Error rate of RNAP?
10-4 per nt
Does RNAP need primers like DNAP does?
no. because transcription need not to be as accurate as replication
Why doesn’t transcription need to be as accurate as DNA replication?
- RNA molecules are temporary, they do not store genetic infromation as long as DNA does
proofreading mechanism of RNAP?
when an incorrect base is added, RNAP can back up, perform excision reaction: reverse polymerisation using water, releasing nucleoside monophosphate
(bacterial) What is a σ factor?
- detachable subunit of the bacterial RNAP
- adheres to consensus sequences on DNA that preceeds pomoter regions
- bacterial RNAP binds to DNA via σ factor
- σ factor together with core enzyme (apoemzyme) are known as the RNAP holoenzyme
(bacterial) What are consensus sequences?
- consensus sequences are optimal seuqences for binding of σ factor
- promoters with consensus sequences in their vicinity initiates transcription much more frequently
what are promoters?
- specific sequecnes that initiates transcription
- A-T rich
inititation of trasncription in both eukaryotes and prokaryotes is described as a stochastic process, what does that mean?
- RNAP sticks weakly to DNA when they randomly collide and slides along it.
- any sequence will occasionally be transcribed => cell has lots of spurious RNA
- RNAP attaches tightly when it encounters a promoter
summary of inititation of transcription in prokaryotes
- RNAP holoenzyme assembles (RNAP core enzyme plus σ factor)
- σ factor adheres to consensus sequences
- RNAP holoenzyme slides along DNA, encounters promoter => binds tightly => starts trancription
What are the three types of eukaryotic RNAP and their respective functions?
- RNAP-I => rRNA
- RNAP-II => mRNA
- RNAP-III => tRNA
(eukaryotes) What are basal transcription factors
- proteins responsible for trancription initiation
- binds to TATA sequences that preceeds promoters
- eukaryotic RNAP binds to DNA via basal transcirption factors
(eukaryotes) what are the main basal transcription factors?
- TFIID
- TFIIB
- TFIIH
(eukaryotes) function of TFIID?
- has TBP (TATA binding protein) which binds to TATA box
- causes large distortion in TATA box
- Brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly
What is TATA box?
- short DNA sequence primarily composed of T and A nucleotides
- a type of promoter sequence
(eukaryotes) function of TFIIB?
- binds to BRE (B response element)
- initiates binding of other trancription factors
- positions RNAP-II
function of TFIIH?
- unwinds and opens DNA strands (helicase activity)
- phosphorylates CTD (C terminal domain) of RNAP-II, this kick starts transcription
summary of eukaryotic initiation of transcription on purified DNA?
- TFIID binds TATA box via TBP
- TFIIB binds BRE
- RNAP-II recruited
- TFIIH opens DNA
- TFIIH phosphorylates CTD of RNAP-II, starts transcription
- basal transcription factors fall off
DNA in eukaryotic cells is packaged into nucleosomes and higher order chromatin structures. Therefore transcription initiation in eukaryotic cell requires more proteins than it does on purified DNA. What are the additional proteins needed?
- activator protein
- mediator protein complex
- chromatin modifying enzymes including:
- chromatin remodeling complexes
- histone modifying enzymes
What is the function of activator proteins
- bind to specific sequences in DNA and help to attract RNAP-II, basal transcription factors and mediator complex to assmeble at promoter
- typical eukaryotic genes have many activator proteins
- sometimes acting from several thousand nt pairs away from gene
What is the function of mediator protein complex?
allows the activator proteins to communicate properly with RNAP-II and with the general transcription factors
what is the function of chomatin modifying enzymes?
- allow access of condensed DNA
- Histone acetyltransferase (a histone modifying enzyme) acetylates lysine and loosens histone binding
How does transcription elongation start?
2 stages before elongation starts:
- initial RNA synthesis (abortive initiation)
- promoter clearance
what is abortive initiation?
- RNAP synthesise about 10 nucleotides rather inefficiently
- depending on how well RNAP is sticking to DNA, it either falls off or start promoter clearance (if sticking well)
what is promoter clearance?
- RNAP conformational change: flap traps RNA, clamp traps DNA), allows it to break away from promoter
- dissociates its σ factor (bacterial)
- disassembly of basal transcription factors (eukaryotes)
- RNAP then starts elongation, synethsising RNA processively
transcription termination in eukaryotes?
polyadenylation: addition of A to the end of mRNA, a post-translational modification
transcription termination in prokaryotes?
- The intrinsic termination reaction
- Rho-dependent transcription termination
What is intrinsic termination reaction?
-
secondary mRNA structure: hairpin
- coded in the DNA template => when transcribed into mRNA, mRNA folds into secondary structure
- hairpin interferes the flap of RNAP, opens flap
- Long pause allows complete formation and separation
what is Rho-mediated termination?
- Rho/ρ factor is a hexameric RNA-DNA helicase that binds to a “loading site” rich in cytosine
- Unwinds the RNA-DNA hybrid causing termination
