molec exam 3 10-13 Flashcards
There are 2 RNA polym. inside of eukaryotic nuclei:
-one to transcribe major ribosomal RNA genes
-one or more to transcribe the rest of the nuclear genes
Why are ribosomal genes different from other nuclear genes?
-different bases/nucleotides than nuclear genes
-unusually repetitive
-found in the nucleolus
Eukaryotic nuclei contain ___ RNA polymerases that can be seperated by _____
3, ion exchange chromatography
RNA polymerase 1
found in nucleolus, likely to transcribe rRNA genes
RNA polymerase 2 and 3
found in nucleoplasm
micro RNA profile …
changes from person to person based on epigenetics, or a persons environment
RNA polymerase 1 synthesizes
large rRNA precursor, for 28s, 18s, 5.8s rRNAs
RNA polymerase 2 synthesizes
hnRNAS (form mRNA), snRNAs, miRNAS (micro)
RNA polymerase 3 synthesizes the precursor for…
tRNA
RPB is …
RNA polymerase 2
RNA poly 2 has __ subunits
12
RPB _ and _ always function together
4 and 7
yeast gene orthologs
RPB1, RPB2, RPB3
RPB1
beta’ ortholog
RPB2
beta ortholog
RPB3
alpha ortholog
what is hRPB1,2,3…
the human version of yeast RPB
Which are the common RPB subunits, common in all three polymerases?
hRPB 5, 6, 8, 10, 12
-little is known about their function.
-likely help transcription process.
Epitome tagging purpose
helps determine whether a polypeptide is a subunit or co purify with the polymerase.
Epitome tagging mechanism
-add extra domain to ONE subunit of RNA poly, with other subunits not changing
-immuno-precipitate with antibody directed against epitope
-denature with SDS detergent, and seperate via electrophoretic gel
RPB’s _______ are REQUIRED for enzyme activity
1,2,3
RPB 1, 2, 3
-homologous to b, b’, and a subunits
-RPB 1 and b’: these bind DNA
-RPB2 and b: at or near the nucleotide joining active site
What are the similarities between RPB3 and alpha subunit?
-a 20 AA subunit very similar
-2 subunits are the same size w/ same stoich.
-2 monomers per holoenzyme
-so, RPB3 is homologous to alpha
What are the two forms of RPB1?
-one can bind to the promote (llb)
-one can transcribe and elongate (llo)
RPB1 gene product is subunit ___
lla.
-lla is the primary product in yeast
-lla is converted to llb by proteolytic removal of the CTD
-converts to llo by phosphorylating 2 serine in the repeating heptad of CTD
-enzyme with lla binds to the promoter
-enzyme with llo is involved in the transcript elongation
CTD
carboxyl terminal domain.
-7 peptide repeated over and over
Which AA’s undergo phosphorylation>
serine
threorine
tyrosine
RPB 4 and 7 help ________
-initiation.
-with rpb 4 and 7, clamp is forced shut
-initiation occurs w the clamp shut, and promoter DNA melts to let template DNA enter active site
-rpb 4 and 7 extend dock region of poly, making it easier for transcription factors to bind
RPB 7
binds to nascent RNA, directing it to CTD
The three RNA polys differ by :
different structures
transcribe different genes
recognize different promoters
RNA poly 2 recognizes class ___ promoters
- Contains:
core promoter- attracts gtf’s and rna poly at basal level, sets transcript start site, and direction of transcription
proximal promoter- enhances transcription. helps attract gtf’s and rna poly, and includes promoter elements upstream of the transcription start site
Core promoter is modular- it can contain any combo of THESE elements:
-TATA box (31 to 36)
-TFIIB recognition (BRE, 37 to 32)
-Initiator (INR, 2 to 4, overlaps w transcript start site)
-downstream promotor element (DPE, 28 to 32)
-downstream core element (DCE, 3 parts)
-motif ten element (MTE 18, to 27)
TATA box
-TATAAA
-similar to prokaryotic 10 boc, centered at -28
-only found in specialized genes, like keratin skin cells or hemoglobin
-promoters are found w no TATA box
TATA-less promoters
-have DPEs
-and are found in 2 classes of genes:
a) housekeeping genes: active in all cells, they control common biochem pathways
b) developmentally regulated genes: control development of fruit fly or immune system in mammals
upstream promoter elements
-found upstream of class 2 core promoter elements
-differ from core promoters in binding to gene-specific transcription factors
-UPE can be orientation dependent, and position dependent- have to be close by.
enhancers and silencers
-position and orientation dependent DNA elements
-rely on tissue specific DNA binding proteins for their activities
-some DNA elements can act either as enhancer or silencer, depending on what is bound to it: like thyroid hormone response element> when bound , enhancer. when not bound, silencer
enhancers
act through the proteins bound to them.
-these proteins are either transcription factors, enhancer binding proteins, or activators
-proteins appear to STIM transcription by interacting with general transcription factors at the promoter
-promote pre-initation complex formation
-usually found upstream of the promoter they control
silencers
act at a distance just like the enhancers to modulate transcription
-INHIBITS transcription
-cause chromatin to coil up, into an inaccessible form to prevent further transcription
eukaryotic rna poly
-INCAPABLE of binding to promoters by themselves
-rely on transcription factors to direct them:
general transcription factors and gene specific transcription factors (AKA activators)
GTF’s combine with rna poly to form a _______ complex.
pre-initiation
-initiates transcript when nucleotides are available
-tight binding forms an open promoter complex with DNA at the transcript start site that has melted
Class 2 preinitation complex
contains:
-rna poly 2, six gtf’s:
TFII A, B, D, E, F, H
-these tf’s and poly form the preinitiation complex in a specific order
Transcription factors bind to class 2 promoters in this order:
-DA complex: TFIID w help from TFIIA binds to TATA box
-DAB complex: TFIIB binds DA complex.
-DABPolF complex: TFIIF helps RNA poly bind to a region from 34 to 17
-DABPolFEH complex: TFIIE and then TFIIH bind to form complete preinitiation complex
TFIID contains these subunits:
TATA box binding protein (TBP)
-evolutionarily conserved, binds to minor groove of TATA box. saddle shaped TBP lines up w DNA, bottom of saddle forces minor groove open, TATA box is now bent
TBP associated factors (TAFs)
specific for Class2. occurs when theres no TATA box.
TBP (tata binding) mutant cell extracts are deficient in …
TBP is a ….
-transcript of class 1, 2, and 3 genes
-universal transcription factor required by poly 1, 2, and 3
TBP associated factors (TAFs)
-13 TAFs are associated w class 2 preiniation complexes
-named according to size, large to small
-interact w core promoter elements and gene specific transcription factors
-when TBP is attached, it will extend the binding of TFIID beyond the TATA box
Roles of TAF’s:
TAF1: helps TFIID bind to initiator
TAF1 has 2 enzymatic activities: histone acetyltransferase and protein kinase
TAF2: helps TFIID bind to DPE
TAF’s enable TBP to bind to TATA-less promoters, that have elements like a GC box.
-different combos of TAF’s is needed for responding to different activators
TFIIH
last general transcription factor to join preinitiation complex
-it contains 9 subunits
-2 major roles in transcription initiaition:
-phosphorylates CTD of RNA poly 2
-unwinds DNA at transcript start site to create transcription bubble
phosphorylation of the CTD of RNA poly 2
-preinitiation complex is formed w hypo phosphorylated RNA poly 2 (lla)
-TFIIH phophorylates serines 2 and 5 in the heptad repeats (YS2PTS5PS7) in the CTD of the largest RNA poly subunit:
creates the phosphorylated form of the poly enzyme llo, phosphorylation at this site is essential for initiation of transcript
minimal initiation complex
TFIID, TFIIB, TFIIF, and RNA poly2 form minimal initiation complex at initiator site
-when TFIIH, TFIIE, and ATP are added they allow DNA melting and partial phosphorylation of the CTD of the largest RNA poly subunit
^^^these events allow abortive transcripts to be made, as the transcription stalls at 10.
expansion of transcription bubble
-energy is provided by ATP
-DNA helicase of TFIIH unwinds DNA
-expansion of the transcription bubble releases stalled polymerase, polymerase clears promoter
elongation complex continues elongating the RNA synthesis when
polymerase CTD is further phosphorylated by TEFb, NTPs are continuously available
-TBP and TFIIB remain at the promoter
-TFIIE and TFIIH are not needed for elongation, and leave the elongation complex
TFIIS
-TFIIS stims proofreading
-TFIIS stims RNase activity of the RNA poly, allowing RNA poly to cleave off an incorrect nucleotide and replace it.
___________ dictate the starting point and direction of transcription
General TF’s.
-by themselves, they will create low level of transcript/basal transcription.
-transcription of many active genes in cells rises above the basal level
-euk cells have extra gene specific TF’s- the activators.
activators bind to enhancers to boost transcription.
activators can _____ or ______ transcription by RNA poly 2
stim or inhibit.
-activators are made of at least 2 domains:
-dna binding domain
-transcription activation domain
-dimerization domain
protein domain
independently folded region of protein
-have DNA binding motif
-part of the domain has shape specialized for DNA binding
-most motifs fall into 3 classes: zinc containing modules, homeodomains, bZIP and bHLH
zinc containing modules
3 kinds, they act as DNA binding motifs:
zinc fingers, zinc modules, modules with 2 zinc ions and 6 cysteines
-all three use one or more Zinc ions to create a shape to fit an alpha helix of the motif into the DNA major groove
Homeodomains
-contain 60 AA’s
-helix turn helix structure/function
-found in a variety of activators
-identified in homeobox proteins for fruit fly development
bZIP and bHLH motifs
-highly basic DNA binding motif linked to protein dimerization motifs
-leucine zippers
-helix loop helix
transcription activating domains
activators have one of these domains, some have more than one:
-acidic domains
-glutamine rich domains
-proline rich domains
DNA binding motifs of activators: structure
-well defined structures
-x ray crystallog show how these interact w DNA targets
-interaction domains can form dimers or tetramers
-most DNA binding proteins can’t bind DNA in monomer form
Three Zinc fingers in a Curved shape
zinc finger is made of:
anti parallel beta strand with
2 cysteines, 2 histidines in alpha helix, helix and beta strand are coordinated to a zinc ion
A third class of zinc modules are found in ________
nuclear receptors.
nuclear receptors are proteins that interact with a variety of endocrine signaling molecules.
-protein+endocrine molecule = complex that functions as an activator, binding to hormone response elements
Type 1 Nuclear receptors
-reside in cytoplasm, bound to another protein.
when receptors bind to their hormone ligands:
-they release their cytoplasmic protein partners, move to nucleus, bind to enhancers, act as activators
Glucocorticoid Receptors: type 1 Nuclear Receptor
-DNA binding domain with 2 zinc containing modules
-one module has most DNA binding residues, the other has surface for protein protein interaction to form dimers
Type 2 and 3 Nuclear Receptors
-Type2 nuclear receptors stay in the nucleus bound to target DNA sites, like thyroid hormone receptors
-without ligands, the receptors repress gene activity, when receptors bind ligands, they activate transcription
-type 3 receptor ligands are not identified “orphan”
homeodomain DNA complex
-proteins are members of the helix turn helix family of DNA binding proteins
-each homeodomain has 3 alpha helices, 2nd and 3rd forming the helix turn helix motif
-a recognition helix fits into the DNA major groove and makes specific contacts there.
-N terminal arm goes into adjacent minor groove
bZIP domains
- proteins dimerize through a leucine zippper
-puts adjacent basic regions of each monomer in position to embrace DNA target like tongs
bHLH domains
-proteins dimerize through a helix loop helix motif, allows basic parts of each long helix to grasp the DNA target
-bHLH and bHLH-ZIP domains bind to DNA in the same way, but bHLH-ZIP have extra dimerization due to leucine zippers
Bacterial core RNA polymerase is….
incapable of initiating meaningful transcription
RNA poly holoenzyme can…
catalyze basal level transcription
-insufficient at weak promoters
-cells have activators to boost basal transcript to higher level in recruitment process.
eukaryotic activators recruit RNA poly to the …
promoters.
-they stimulate binding of the GTF’s and RNA poly.
the hypothesis:
-gtf’s cause stepwise build up of preinitiation complex, or gtf’s + proteins are already bound to poly, in a RNA poly holoenzyme
__________ must interact to form the preinitiation complex
GTF’s
Activators and ______ interact
GTF’s
activators usually interact with one another in activating a gene
-individual factors interact to form a protein dimer helping binding to a single DNA target
-specific factors bound to different DNA target sites can collaborate in activating a gene
dimerization
-increases affinity between activator and its DNA target
-some activators form homodimers but others function as heterodimers
insulators
-can shield genes from activation by enhancers, enhancer blocks activity
-can shield genes from repression by silencers, barrier activity
regulation of transcription factors:
-phosphorylation
-ubiquitylation
-sumoylation
-methylation/acetylation
phosphorylation
phosph. of activators can allow them to interact with coactivtors, to stim transcript
ubiquitylation
ubiquitylation of tf’s can mark them for destruction by proteolysis and for stimulation of activity
-mono-ubiquitylation especially of some activators can have an activation effect
-poly-ubiquitylation marks these proteins for destruction
-proteins from the 19s regulatory particle of proteasome can stim transcript
sumoylation
attachment of polypeptide SUMO which can target incorporation into compartments of the nucleus
-SUMO=small ubiquitin related modifier
-101 AA polypeptide
-sumoylation is the addition of one or more copies of SUMO to lysine residues on a protein, process similar to ubiquitylation, but results are different.
-sumoylated activators are targeted to a specific nuclear compartment that keeps them stable
methylation/acetylation
modulate activity
acetylation
non histone activators and repressors can be acetylated by histone acetyltransferase (HAT)
-HAT can act on non histone activators and repressors
-can have either pos or neg effects
Eukaryotic genes can exist as naked DNA/or DNA molecules only bound to tf’s (t/f)
false
Chromatin
DNA + protein to form chromosomes
-chromatin is variable, and the different versions play an enormous role in chromatin structure/the control of gene expression
euk cells have 5 kinds of histones:
H1, H2A, H2B, H3, H4
histone proteins are not homogenous because…
gene reiteration, and posttranslational modification
histone structure
-abundant proteins whose mass in nuclei nearly equals that of DNA
-positive charge at neutral pH
-well conserved from one species to another
-not single copy-they are repeated many times.
-some copies are identical, some vary
-H4 has only 2 variants ever reported
post translational modification of histones
-most common mod is acetylation
-at N terminal amino groups and lysine E amino groups
-methylation and phosphorylation also take place at lysine E amino groups
-serine and threonine undergo O phosphorylation
-histone modifications influence chromatin structure and function, and play important role in gene activity
nucleosomes
chromasomes are long and thin molecules, they will tangle if not properly folded. folding occurs in a few ways, the first order of folding is the Nucleosome.
-nucleosomes have core of histones, and DNA winds around it.
-X ray diffrac has shown strong repeats of structure at 100A intervals
-this corresponds to a string of nucleosome about 110A in diameter
histones in nucleosome
chemical cross linking in soln:
H3 - H4
H2A - H2B
H3 and H4 exist as a tetramer.
corresponds to 1 histone octamer per 200 bp of DNA
Octamer is composed of 2 each H2A, H2B, H3, H4
1 each H1 bound to octamer
chromatin is made of
roughly equal mass of DNA and histones.
H1 and chromatin
treatment of chromatin with trypsin or high salt buffer removes histone H1, leaving chromatin like beads on a string
the beads are nucleosomes.
core histones form a ball with DNA wrapped on outside, DNA on outside minimizes amount of DNA bending.
H1 lies on the outside of the nucleosome
Nucleosome structure
central H3 H4 core attached to H2A H2B dimers
-grooves on surface define a left hand helical ramp, the path for DNA winding
-DNA winds almost twice around the histone core condensing DNA length by 6 to 7 x.
-core histones contain histone fold
histone fold
3 alpha helices linked by 2 loops
extended tail of about 28 % core histone mass
tails are unstructured
second order of chromatin folding produces a 30nm fiber
string of nucleosomes condenses to form the 30nm fiber in a soln of increasing ionic strength
-this condensation results in another 6 to 7 fold condensation of the nucleosome itself
4 nucleosomes condensing into the 30nm fiber form a zig zag
solenoid model
hollow compact helix
high order chromatin folding
30nm fibers account for most of the chromatin in a typical interphase nucleus
further folding is needed in structures like the mitotic chromosomes
the series of radial loops is a model known for high order folding
histones, especially H1, have a _____ effect on gene activity
repressive
histones play a predominant role as _____ of ______ and are not just structural
regulators, genetic activity
histone H1 causes _______ of template activity and core histones
further repression
H1 repression can be…
counteracted by tf’s
Sp1 and GAL4:
anti repressors, prevent histone repression, as well as they can act as transcription activators
transcription factors can cause anti repression by:
-removing nucleosomes that obscure promoter
-preventing initial nucleosome binding to promoter
^^both actions are forms of nucleosome positioning, activators force nucleosomes to take up positions around promoters
histone acetylation
occurs in cytoplasm and in nucleus.
cytoplasmic acetylation carried out by histone acetyltransferase B (HAT-B):
prepares histones for incorporation into nucleosomes, and acetyl groups later removed in nucleus
nuclear acetylation
-catalyzed by HAT-A
-acetylates core histone N terminal tails
-correlates with transcription activation
-coactivators of HAT-A found, may loosen nucleosomes with gene control region
-attracts bromo domain proteins, essential for transcription
histone deacetylation
transcription repressors: bind to DNA sites and interact w co repressors which bind to histone deacetylases
retinoic acid receptor: binds to retinoic acid response elements (RARE) as a heterodimer with RXR (retinoid x receptor)
in the absence of RA, the RAR-RXR heterodimer inhibits transcription through recruitment of histone deacetylases and nuclear receptor corepressors
In the presence of RA, the RAR-RXR heterodimer
leaves the HDAC complex and recruits transcriptional coactivators
HDAC in transcription repression
-assembly of complex brings histone deacetylases close to nucleosomes
-deacetylation of core histones allows histone basic tails to bind to DNA and histones in nearby nucleosomes to inhibit transcription
Heterochromatin
euchromatin:
extended and open chromatin, potentially active
heterochromatin:
very condensed, DNA is inaccessible
-microscopically appears as clumps in higher eukaryotes
-repressive ability able to silence genes even 3kb away.
