Lecture 18 Flashcards
How does eukaryotic and prokaryotic transcription differ?
- Eukaryotes have a nucleus so transcription and translation are not coupled
- Eukaryotes have chromatin - the transcription machinery must work around nucleosomes. (DNAse I “hypersensitivity” is used to identify transcriptionally activeregions of interphase chromatin – identifies regions that are not protected byregularly-spaced nucleosomes.)
- Eukaryotes have more complex processing of mRNAs:5’ capping, 3’ poly A tails, splicing out of intron sequences
How many different RNA polymerases (RNAPs) do Eukaryotes have?
3
Fxn of RNA pol I
(RNAP I) synthesizes rRNA ( 18S, 5.8S, and 28S) ➞ part of ribosomes
Fxn of RNA pol 2
synthesizes mRNA ➞ proteins
Fxn of RNA pol 3
synthesizes tRNA (adaptor for protein synthesis), 5S RNA (part of ribosomes)
How much of total RNA is rRNA, tRNA, mRNA?
~80% of the total RNAin rapidly growing mammalian cells is rRNA and 15% is tRNA;protein-coding mRNA constitutes only a smallportion of the total RNA but is very diverse.
Where are RNAP I, II, III found?
- I is in nucleolus
- II and III is in nucleoplasm
Effects of alpha-amanitin on RNAP I,III, III?
- I is insensitive
- II is strongly inhibited
- III is inhibited by high concentrations
Effect of alpha-amanitin on RNAP II during transcription?
arrests RNAP II
during the elongation phase of transcription.
How is using alpha-amanitin useful?
can be used to determinewhich RNA polymerase is responsible for transcribing a given RNA in vivo
What do eukaryotic RNAPs need in order to bind DNA promoters?
Transcription factors
Role of various TFs?
- Some bind DNA and help RNA pol to bind (i.e., they stabilize RNA polymerase binding, like CRP in prokaryotes);
- Some enhance transcription via protein-protein interactions
- Different TFs often act together to influence transcription.
What does it mean that TFs are species-specific?
recognize
promoters in their
own species
In eukaryotes, what provides specificity? How does this differ compared to prokaryotes?
TFs, rather than the
RNAPs, provide
specificity for a given
promoter
What is the nucleolus?
a large non-membrane bound structure within the nucleus that synthesizes and assembles ribosomes
How many subunits does RNA pol I have? How big is it?
- large complex, contains 13 subunits, 600 kDa
How many ribosomes does RNA pol I need to make for each cell generation?
10,000,000
All 3 rRNAs (18S, 5.8S, 28S) are transcribed as what first?
- transcribed as a single “45S” precursor RNA
When is 45S cleaved and where?
during ribosome
assembly - multi-step processing
- occurs in
the nucleolus
How many subunits does RNA III have? How big is it?
- largest, 700 kDa
- most complex of the RNAPs: 14 subunits
How many 5S rRNA gene in cell?
500-20,000 copies
What do 5S rRNA and all tRNAs have in common?
small, do not encode proteins,and have unique regulatory sequences
What rRNA makes up 60S subunit of ribosome?
- 5S, 5.8S, 28S
All cellular proteins are derived from?
- mRNAs
Initially mRNA in eukaryotes are transcribed as what?
- hnRNA (heterogenous nuclear RNA) aka pre-mRNA aka primary RNA transcript
- unprocessed form of mRNA
pre-mRNA needs to have what done before it becomes mRNA?
- 5’ capping, intron splicing, 3’poly-adenylation
Eukaryotic and prokaryotic transcribed as monocistronic or polycistronic msgs?
- Eukaryotic = polycistonic; eukaryotic cells are generally not organized into operons: one promoter/one gene
- Prokaryotic cells are organized into operons (one promoter for many genes)
Steps to eukaryotic transcription?
- The TFIID complex binds to the TATA box via the TATA binding protein (TBP)
- Binding of TFIID enables TFIIA and TFIIB to bind.
- RNAP II and additional transcription factors, some of which are already associated with RNAP II, bind to the DNA-TF complex
-> closed complex. TFIIH is an ATP-dependent helicase that unwinds
the DNA at the Inr site for polymerization -> open complex. - TFIIH also phosphorylates the C-terminal domain (CTD) of RNAP II, which then clears the promoter and polymerization is initiated, with most of the TFs falling off. Phosphorylation regulates additional
factors (e.g., 5’-capping, splicing enzymes) binding to the CTD.
RNAP II-mediated transcription is normally regulated by?
2 control elements in the DNA:
- “promoter–proximal” are upstream (5’) of and close to the gene to be transcribed
- enhancer elements: DNA sequences distant from the promoter and gene
Promotor-proximal regions contain what?
- TATA box at -25 position (a core promoter element) with consensus sequence TATA(A)/(T)A
- CAAT box (consensus GCCCAATCT) 100-200 bp upstream of the transcription start site
First step in formation of transcription complex?
TATA box is bound by the RNAP II TF, TFIID via its TATA binding protein (TBP)
Where is DNA unwound?
- Initiator (Inr) sequence at +1 (transcription start site)
How many TFs bind to RNAP II promotor region and why?
multiple TFs bind to RNAP II promoter region and together affect the level of gene
expression, resulting in coordinated and effective regulation of transcription
Fxn of enhancer elements?
- not part of promotor (far away)
- proteins bind to enhancers and to proteins bound at the promoter, linking the two distant sequences
Fxn fo TFIIH?
- an ATP-dependent helicase that unwinds
the DNA at the Inr site for polymerization -> open complex - phosphorylates the C-terminal domain (CTD) of RNAP II, which then clears the promoter and polymerization is initiated, with most of the TFs falling off
What TF initiates polymerase complex?
TFIID
How does TFIID bind to TATA box?
via its TATA binding protein, TBP, a 30 kDa component of the large (700 kDa) TFIID complex –TBP is a saddle-shaped protein containing two similar domains
TBP of TFIID binds to where on TATA box
TBP binds to the minor groove of the TATA box via its concave surface
What does the biding of TBP of TFIID cause
- binding induces a large conformational change in the DNA, bending it and spreading the minor groove
- Phe side chains of TBP intercalate between the base pairs of the minor
groove
the unique conformation of the TFIID and the kinked DNA may provide
a dockingsite for additional TFs and RNAP II
How do eukaryotic genomes differ from prokaryotic genomes
- Unlike prokaryotic genomes, which use
both positive and negative regulation to activate and/or repress transcription, eukaryotic genomes are mostly positively regulated - Initiation of transcription almost always requires the action of multiple activator
proteins
Why is eukaryotic genomes positively regulated and not negatively regulated?
The presence of chromatin means that most promoters are inaccessible to polymerases - this acts as a type of repression, so negative regulation by protein repressors is not necessary
What does the presence of multiple activator sequences ensure in eukaryotic transcription?
genes are not activated
non-specifically - with ~20,000 genes in the human genome, the chances of an activator sequence being present in many sites is very high, but the presence of multiple activator sequences ensures specificity of transcription initiation.
In order for transcription to proceed, an active RNAP II complex must form at the
promoter (i.e., RNAP II plus the TFIIs). This requires the 2 actions:?
1) histone modifying enzymes and chromatin remodeling proteins to exposed the
DNA to be transcribed
(2) regulatory proteins to promote transcription.
What are activators?
- bind to enhancer elements on the DNA and enhance transcription levels for specific genes (note that activators are sometimes also referred to as TFs)
- can help in the assembly of the polymerase initiation complex by helping to recruit RNAP II to the promoter
What are enhancers and where are they found?
regulatory DNA sequences that can operate thousands of base pairs (up to
50,000) away from the promoter and +1 site of a gene – they can even be downstream of the
gene
Fxn of enhancers?
- bind multiple regulatory proteins (transcription activators), including some that also bind at the promoter, linking the promoter and the enhancer
- increase transcription at nearby promoters regardless of their orientation in the DNA
An average RNAP II promoter is affected by how many enhancers?
Average half a dozen
What are some regulatory proteins?
transcription activators, chromatin modification and remodeling proteins, co-activators and transcription factors (TFIIs)
Enhancer-bound activators can be liked to the promotor bound by what two ways?
- Linked to promotor-bound TFs directly
- Linked to promotor-bound TFs via “co-activators”
What is a mediator? Fxn?
- Large complex formed from multiple co-activators
- Acts as intermediary between activators and the RNAP II complex
- Allow activators to communicate w/RNAP II and other TFs
- additional complexes can interact with the mediator and inhibit transcription
What modulates activators?
Molecules/ligands i.e. cell can “sense” changes in environment and respond by changing expression of select genes (think effectors influencing activators in prokaryotic transcription)
What is needed to access DNA (chromatin) for eukaryotic transcription?
chromatin remodeling complexes and histone acetylases are also necessary to move nucleosomes around and allow the TFs, polymerases and activators access to the DNA
In chromatin remodelling, what is TAP?
activator protein
(transcription activator protein – binds to an enhancer sequence in
DNA)
In chromatin remodelling, what is CRC?
is the chromatin remodelling complex
In chromatin remodelling, what is TBP binding site?
TATA box
In chromatin remodelling, what is TBP?
TATA-box binding protein
In chromatin remodelling, what is TBP?
TATA-box binding protein
What is added at 5’ end of mRNA during transcription?
specialized structure (a “cap”)
How does the 5’ cap initiate translation?
cap interacts w/ ribosomes to initiate
translation
How is 5’ cap formed?
- formed by condensation of a molecule of GTP w/ triphosphate at 5’ end of hnRNA in an unusual 5’-5’-triphosphate linkage
- the guanine is subsequently methylated atN-7 ® 7-methylguanosine (7-Methyl G)
During capping where are methyl groups often added?
- often added to the ribose 2’-OH of the first and second nucleotides
adjacent to the cap - if the first nucleoside is adenosine
(it is usually a purine), it may also be N6-
methylated
capping (and methylating) enzymes areassociated with what of RNAP II
C-terminal domain
CTD
When is 5’ cap added?
early on, before synthesis of the primary mRNA transcript is complete
Protective fxns of 5’ cap?
may also stabilize mRNA by protecting it from nucleases
Which mRNA processing enzymes are associated with the CTD of RNA polymerase II
- capping enzymes
- enzymes/factors involved
in splicing - polyadenylation enzymes
different processing
enzymes bind to the CTD depending on
- its phosphorylation state
What does the cleavage signal sequence include?
- Highly conserved AAUAAA sequence
When does RNAP II stop synthesizing RNA?
Beyond cleavage signal sequence
3 steps in polyadenylation?
- a protein complex that includes an endonuclease, polyadenylate polymerase, and several other proteins binds to both the cleavage signal
sequence in the mRNA, and the CTD of RNAP II - the endonuclease cleaves the transcript 10–30 bases downstream from (i.e., 3’ to) AAUAAA, releasing it from RNAP II
- the polyadenylate polymerase adds
80-250 A residues
The 3’ polyA is important for mRNA export from the nucleus, protein translation (associating with ribosomes)
and stability (protects mRNA from nucleases).
During polyadenylation, what binds to both cleavage signal sequence and CTD of RNAP II?
protein complex that includes an endonuclease, polyadenylate polymerase, and several other proteins
During polyadenylation where does the endonuclease cleave
the transcript 10–30 bases downstream from
(i.e., 3’ to) AAUAAA, releasing it from
RNAP II
Why is 3’polyA important?
- for mRNA export from the nucleus
- protein translation (associating with ribosomes)
- stability (protects mRNA from nucleases).
What are zinc finger proteins
important class of transcriptional regulators
What do zinc finger domains consist of?
~30 amino acids long, with an a-helix and a loop or b-hairpin stabilized by a Zn2+ ion, which is coordinated by 4 cysteines or 2 cysteines and 2 histidine side chains
Where do zing finger domains bind to
Major groove of DNA
Example of DNA binding protein that uses zinc finger domains
TFIIIA (transcription factor A for RNAP III) binds to the 5S RNA promoter via zinc
finger domains
The homeodomain is folded into what? What holds the structure together?
Folded into 3 alpha-helices packed tightly together by hydrophobic interactions
Helix 2 and 3 in the homeodomain resemble what?
The helix-turn-helix motif
What is the recognition helix? What does it interact with?
Helix 3, interacts specifically w/bases in the major groove
What do the AA’s in helix 1 interact with?
Baise pairs in the minor groove
What is the leucine zipper? How formed?
- Coiled coil
- formed by two alpha-helices that dimerize thru hydrophobic interactions btwn leucine side chains spaced every 7 AA’s
What do the recognition helices in the leucine zipper interact with?
The DNA in the major grooves on either side of the DNA
Are dimers in the leucine zipper homologous (identical a-helices) or
heterologous (2 different a-helices)?
They can be both
