Transcription I Flashcards
What are the differences btw RNA and DNA?
RNA
-
D-ribose instead of 2’-deoxyribose
→ can be hydrolyzed by alkalis to 2’, 3’ cyclic diesters due to presence of 2’-OH group - uracil instead of thymine
- usually single strand instead of double strand (except in certain viruses)
List different types of RNA.
protein coding RNAs
- mRNA: templates for protein synthesis
nonprotein coding RNAs
- rRNA: form ribosomes
- tRNA: adapter molecules for translation
- small RNA: see own card
- ribozymes: catalyticly active
List different types of small RNA.
- snRNA
- snoRNA
- miRNA
- siRNA
- piwiRNA
What is snRNA?
Where can it be found + function?
small nuclear RNA
- in eukaryotes, archae
- involved in splicing of pre-mRNA
What is snoRNA?
Where can it be found + function?
small nucleolar RNA
- in eukaryotes, archae
- involved in chemical modification of rRNA/tRNA/snRNA
What is miRNA?
Where can it be found + function?
micro RNA
- in eukaryotes
- inhibition of translation of mRNAs
What is siRNA?
Where can it be found + function?
small interfering/silencing RNA
- in eukaryotes
- cause degradation of RNA molecules due to hybridization (= silencing)
What is piwiRNA?
Where can it be found + function?
Piwi-interacting RNA
- in animals
- protection against transposons
What are differences btw RNA and DNA synthesis, hence transcription and DNA replication?
in RNA synthesis
- ribonucleotides used
- U instead of T
- no primer necessary, initiation starts de novo
- only portions transcribed, genes can overlap to code for different proteins
- no proofreading → lower fidelity
Differentiate btw coding, non-coding, and template strand.
-
template strand: DNA strand that is transcribed
= non-coding/antisense strand -
coding strand: DNA strand strand which has the same base sequence as the RNA transcript produced (obv T instead of U)
= non-template/sense strand
NOTE: both strands can be used as templates though
What is asymmetric transcription?
both DNA strands could be used as templates, but transcription would be happening in opposite directions (still both in 5’→3’ direction)
just like DNA replication: synthesized in 5’→3’ direction, template read in 3’→5’
Describe the basic mechanism of transcription.
- requires the unwinding of the double helix at gene that should be transcribed
-
RNA polymerase
- catalyzes the formation of phosphodiester bonds between nucleotides
- moves stepwise along the DNA extending the RNA chain as it goes
- as it moves it unwinds the next part of the helix
- helix behind RNA polymerase closes and the mRNA is displaced
What are different features of DNA/RNA polymerase?
RNA polymerase
- has intrinsic helicase activity
- only works on one strand
- does not require priming
- does not proofread
How many types of RNA polymerase (RNAP) are there in prokaryotes?
Describe its structure.
only one type
4 subunits:
- 2α: bind regulatory sequences
- β: forms phosphodiester bond
- β’: binds DNA template and σ factors
What are σ (sigma) factors, what do they do?
DNA-binding proteins, bind to β’ of prokarytoic RNAP to form RNAP holoenzyme Eσ
⇒ allow recognition of promoter
NOTE: σ factor considered an own subunit, so 5 subunits now
Describe the molecular mechanism how RNAP holoenzyme Eσ binds to the DNA.
recognizes atomic pattern in major groove of DNA (depending on base pairing)
- acts as H donor, or acceptor, forming H-bridges
- recognizes methyl groups, hydrophobic interactions
- recognizes H atom, unavailable for H-bonding
What does the +/- numbering system indicate?
- = upstream
- = downstream
⇒ way of defining the location of regulatory elements in a gene
How do you call the site on the DNA template where RNAP holoenzyme Eσ binds?
Structure.
promoter, upstream of transcription start site TSS
- -35 region w/ consensus sequence TTGACA
- 16-19bp spacer
- -10 region, Pribnow box
What is the Pribnow box?
AT-rich sequence in prokaryotic promoters, easier unwinding due to only 2 H-bonds
→ similar to TATA box in eukaryotes
Differentiate btw types of σ factors.
recognize different promoters + activate transcription of different genes
- sigma 70: housekeeping sigma factor (normal one)
- sigma 54: nitrogen-limitation sigma factor
- sigma 38: starvation phase sigma factor, activates gene involved in long term survival (i.e. peroxisomes)
- sigma 32: heat shock sigma factor
NOTE: variety of σ factors + competition for RNAP determines much of cell’s protein content
Which method is used to search for promoters on DNA?
footprinting
What happens after RNAP holoenzyme binds to the promoter?
forms pre-initiation complex
RNAP uses helicase activity to unwind dsDNA at Pribnow box, forming an open promoter complex
⇒ initiates transcription of new 5’ terminal of RNA a bit more downstrean at TSS (transcription start site)
How is the first bond of the new mRNA during initiation of transcription formed?
standard mechanism
RNAP couples first base to second, formation of dinucleotide w/ phosphodiester bond
REMEMBER: strand synthesized in 5’→3’,
hence DNA template strand read in 3’→5’ direction
Describe the elongation of prokaryotic transcription.
- after 8-9 bp of RNA synthesis, σ factor released
= promotor clearance - RNAP completes transcription at 30-50bp/s
- unwinds (pos. supercoils of DNA) + reanneals behind enzyme (forming neg. supercoils)
⇒ RNA-DNA hybrid formed, but most RNA displaced as DNA helix reforms
What are the 2 types of termination of transcription?
Differentiate btw pro- and eukaryotic termination.
(type I) ρ-independent:
- in prokaryotes
- palindromic sequence forms a hair pin loop, destabilizes DNA-RNA hybrid
(type II) ρ-dependent:
- in eukaryotes
- termination protein ρ factor binds to RNA, breaks H-bonds btw template DNA and RNA, then dissociates
→ disrupt elongation complex
How is the prokaryotic rRNA formed?
- synthesized like mRNA. different precursor rRNAs are synthesized
- 30S precursor rRNA cleaved by RNase to 16S rRNA, 5S rRNA (small ribosomal subunit), 23S rRNA (large ribosomal subunit) and selected tRNAs
NOTE: precursor determines which tRNAs are produced
What do Svedberg units correspond to?
= [S] molecular size and shape
What does actinomycin D do?
Which compound has a similar effect?
acridine
- intercalate btw successive G=C base pairs in dsDNA
- inhibit transcriptional elongation in pro- and eukaryotes
What does rifampicin do?
antibiotic
- binds the β subunit of bacterial RNA polymerase
- blocks promoter clearance (elongation)
What are true activation and antirepression?
Where does it occur?
occurs in eukaryotes to activate chromatin for transcription
- DNA in inactive ground state (condensed heterochromatin)
- antirepression: conversion to depressed state (deconsed euchromatin)
- true activation: conversion to activated state
Describe the mechanism of activation of chromatin for transcription.
- transcription factor binds to DNA + recruits histone acetylase (HAT)
- histone acetylase acetylates histones to decr. interactions w/ DNA (ex: H3 lysine9 acetylation)
- chromatin remodeling complex unwraps nucleosomes at the promoter in an ATP-dependent manner
⇒ eventually RNAP holoenzyme bind to initiate transcription
How is transcription regulated in eukaryotes?
binding of trans-acting factors to cis-acting elements on DNA
⇒ controls transcription (= gene expression)
What are cis-acting elements?
Examples.
DNA sequences close to a specific gene, affecting its transcription
- promotors: containing transcription start site
- enhancers: regulatory DNA sequences, bind TFs to upregulate transcription
- silencers: regulatory DNA sequences, reduce/shut off expression of a gene
- terminators: stop signal for transcription
What are the characteristics of enhancers?
What are trans-acting factors?
Examples.
usually proteins (can be hormones, heavy metals) that bind to cis-acting elements to control gene expression
- may be expressed in a spec. tissue
- may be expressed at spec. time in development
- may be required for protein mod.
- may be activated by ligand binding
⇒ RNA polymerases, transcription factors
What are the 2 groups of transcription factors?
Why are they important?
specific + general TFs
- Pol I, Pol III promoters mainly require general TFs
- Pol II promoters mainly require specific TFs
How do transcription factors bind to cis-acting elements on DNA?
via DBD (DNA binding domain)
→ bind through H-bonds/van der Waals interactions to DNA in major groove
- Zinc finger domain
- helix-turn-helix domain
- Leucine zipper domain
- basic helix-loop-helix
Differentiate btw eukaryotic RNAPs and their function.
Where are they located?
3 nuclear RNAPs: Pol I in nucleolus, others in nucleoplasm
- Pol I: transcribes genes coding for 18S, 5.8S, 28S rRNA
- Pol II: transcribes genes coding for mRNA, miRNA, snRNA
- Pol III: transcribe genes coding for tRNA, 5S rRNA
+ 1 mitochondrial RNAP IV
⇒ require transcription factors to assemble at promoter
How are eukaryotic RNAPs structurally different from prokaryotic RNAP?
additional subunits to provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities
What does α-amanitin do?
in Amanita phalloides (death cap mushroom)
potent inhibitor of RNA pol II and weak inhibitor of RNA pol III
NOTE: RNAP I = insensitive to α-amanitin
How is mtDNA transcribed to mRNA?
both strands are transcribed (entirely)
- D loop contains promoter for each strand
- RNA then cut into inidividual RNAs for each gene
- protein-coding genes are poly-A-tailed, r/mRNAs modified as necessary
Explain the initiation of transcription at RNA Pol I promotors.
-
upstream binding factor (UBF) (2 subunits) binds to upstream core element (UCE) and the core promoter element (CPE)
→ protein-protein interactions to bring both closer together - subsequent binding of selectivity factor I (4 subunits)
→ stabilizes structure, RNA pol I binds
UBF somewhat similar to sigma factor in prokaryotes
What is the product of transcription of RNA pol I?
How is it processed?
pre-rRNA transcript 45S
⇒ methylated + cleaved to mature 18S, 5.8S, 28S rRNA
How are snoRNAs transcribed?
- some synthesis by RNA pol II (having snoRNA specific promoters)
-
majority formed from introns
- from genes coding for proteins that are involved in ribosome synthesis/translation
- from genes coding for non-functional mRNAs
Explain the initiation of transcription at RNA Pol III promotors.
What are its transcriptional products?
- transcription factor TFIIIC binds to A and B box on DNA
- trimeric TFIIIB binds immediately upstream of TSS
→ recruits RNA pol III
⇒ synthesizes 5S rRNA and tRNA (as seperate precursor RNAs)
BUT: mechanism for 5S rRNA slightly different
again, here TFIIIC somewhat similar function to sigmar factor in prokaryotes
How is the transcription of 5S rRNA by RNA pol III different fromt that of tRNA?
TFIIIA has to bind to C box, before TFIIIC binds to A and B box
How are tRNAs processed?
- RNase P and D insert cuts in primary transcript
- bases modified and other minor modifications done to form intermediate
- spliced to mature tRNA
What is one of the most frequent naturally occuring post-transcriptionally modified bases in tRNA?
Structure.
pseudouridine
Which DNA region is especially important for the initiation of mRNA transcription by RNA pol II?
Why?
TATA box
located 25 nucleotides upstream from transcription start site, AT-rich sequence for easier unwinding (2 H-bonds)
→ recognized by C-terminal of TBP (TATA-binding protein) of TFIID
How is the initiation of transcription catalyzed by RNA pol II initiated, once TFIID has bound to the TATA box?
- several other TFs and RNA pol III assemble at promoter
-
TFIIH (helicase)
- hydrolyses ATP to unwind DNA at TSS
- phosphorylates RNA pol II
- phosphorylation induces conformational change of RNA pol II → release of TFs, escapes from promotor + starts elongation
At which site is RNA pol II phosphorylated during initiation?
at long C-terminal polypeptide tail, also called the C-terminal domain (CTD)
NOTE: after termination and pol II release dephosphorylated again
What is the function of topoisomerases during the process of transcription?
precede and follow RNA polymerase in transcription bubble → relieve torsional stress
How do you call the primary transcript of RNA pol II?
Where is it synthesized and what happens to it?
heteronuclear RNA (hnRNA) or pre-mRNA synthesized in nucleus
⇒ receives modifications to prepare it for translation
