eukaryote trans 1 Flashcards
who proposed the operon hypothesis?
francois jacob and jacque monod
what are the two classes of genes in an operon?
structural (encoded metabolically related enzymes)
regulatory elements( controlled expression of the structural genes)
Mutations in operon structural genes…
abolished one particular enzymatic activity
mutations in operon regulatory genes…
affected all of the different enzymes under its control
is the lac operon what is the function of lacZ?
b-galactosidase activity, metabolizes lactose
is the lac operon what is the function of lacY?
permease, transports lactose into the cells
what happens is LacZ or Lac Y is mutated?
Lactose transport can still be induced in lacZ mutants, and lacY mutants still display lactose-inducible b-galactosidase activity
what happens if the regulatory region of lac operon is mutated?
no lactose transport and no lactose metabolism
what is the function of lacA?
transacetylase enzymes, transfer an acetyl group from acetyl-CoA to galactosides, lactosides, and glucosides.
what is lacI is mutated?
lacI is the repressor, so you will always have some level of LacZ,Y,A expression
what is LacO function?
the site where lacI (the repressor) binds to repress expression of lac operon
what happens in LacO is mutated?
same as what happens in LacI mutant, expression would be constutive of lac operon
how to distinguish between lacI and lacO mutations?
use meridiploids to add second WT copy of lac operon to e.coli
if LacI isn’t functional in one copy the WT will still make LacI and it’ll bind lacO and repress lac operon transcription (“recessive mutation”)
if LacO isn’t functional in one copy the WT will still be bound by the repressor, but the mutant won’t be bound by the repressor. The mutant would still transcribe lac operon genes (“dominant mutation”)
what is a meridiploid?
e.coli are haploid, but you can add a second copy of a gene introduced into genome or plasmid
define a “cis-dominant” mutation
a mutation which only affects the mutant operon but doesn’t affect the WT operon
define a “trans-dominant” mutation
a mutation which only affects the WT operon but doesn’t affect the mutant operon
what happens if LacI^d is mutated in a meridiploid?
dominant negative repressor protein is produced, lac operon constitutively expressed. a repressor that can no longer bind operator (lacO) is produced, the repressor protein is a tetramer so only one copy being mutated will result in a dysfunctional repressor protein
how many subunits is the lac repressor protein?
tetramer
what happens if LacI^s is mutated in a meridiploid?
this mutant is “unresponsive to the inducer”, cis- and trans dominant
difference between lacI^d and lacI^s mutants?
the s mutant binds the lacO site but is dysfunctional so transcription of lacOperon is not repressed, the d mutant cannot bind the lacO site at all so transcription is not repressed
difference bt prokaryote and eukaryote transcription factor binding?
prokaryotes if the TF is made it will always have access to DNA, in eukaryotes other factors can prevent TF from binding DNA
define nonrestrictive transcription
in prokaryotes TF can always bind DNA
define restrictive transcription
in eukaryotes other factors may need to be present for TF to bind DNA
major difference bt accessing DNA in pro and eukaryotes?
nucleosomes (histones) aren’t present in prokaryotes
reasons there are more oppurtunities to regulate euk transcription than prok?
- separation in time and space of RNA transcription and translation (oppurtunity for post-transcriptional mods)
- multimeric regulatory proteins necessary in euks (cofactors for TF to bind are needed in almost all cases)
how many RNA polymerase genes in bacteria?
1
how many RNA polymerase genes in animals?
3
how many RNA polymerase genes in plants?
5
RNAP means what
RNA polymerase
more RNA polymerase = more ?
more variation in promoter sequences
what is necessary to recruit RNAP for almost all euk genes?
transcription factors
RNAP I?
makes rRNA precursors of most ribosomal genes (28S, 18S, and 5.8S rRNA); functions in the nucleolus
what is the nucleolus?
a site in the nucleus where ribosomal biogenesis takes place, ribo genes are transcribed, processed, and assembled here
RNAP II?
synthesizes all mRNA precursors, miRNA and some snRNAs; functions in the nucleoplasm
snRNA means?
small nuclear RNA
RNAP III?
transcribes all tRNA genes, 5S rRNA gene and some snRNAs; functions both in the nucleolus and in the nucleoplasm
RNAP IV and V (plants only)
synthesize siRNA that are then converted to dsRNA by an RNA-dependent RNA Pol
Plastidic (plants) and mitochondrial RNAPs
synthesize all plastidic and mitochondrial transcripts, respectively
what is a plastid?
A plastid is a membrane-bound organelle found in the cells of plants (chloroplast etc)
what chemical was initially used to determine function of RNAPs?
a-amanitin (found in death cap mushroom)
what does a-amanitin do?
inhibits RNAPII activity
components of ribosomal RNA? what percentage of RNA is rRNA?
18s, 5.8s, 28s
50-90%
differential regulation of gene expression is focused on which RNAP?
RNAPII
what are TFII?
class 2 TFs, work with RNAPII
the majority of RNAPIII genes are?
housekeeping/constitutively expressed
how many RNAPIII subunits?
15
describe naming scheme of RNAP subunits?
Rpa = RNAPI
rpb = RNAPII
rpc = RNAPIII
how many RNAP subunits are common between different RNAP? “core subunits”
4
what RNAPII subunits are similar to the b’ and b subunits of E. coli RNAP?
Rbp1 and Rbp2
how to study RNAP subunits?
tag a subunit gene like Rbp3, then see what interacts with it using co-ip against Rbp3 tag
what is a nonessential subunit of RNAP?
they are only conditionally required, sometimes necessary sometimes dispensable
name the two conditional yeast RNAPII subunits
Rbp4 and Rbp11
crystal structure of RNAPII helped determine subunit function how?
the placement in the structure gives hints of each subunits function
what part of RNAPII structure make up the clamp? what is the clamps functions?
Rbp1, 2, and 6
lock polymerase onto template near catalytic center (allow enzyme to circle DNA and lock, enables processivity)
what part of RNAPII structure make up the pincers/jaws? what is the function?
Rbp1, 5, and 9
grip dsDNA ahead of polymerase
you have to do what to DNA to accomidate it in RNAPII?
bend it
needs local denaturation
what is required for catalytic activity of RNAPII?
magnesium (Mg2+)
how does Mg2+ ion change DNA confirmation?
it binds the phosphate group connecting nucleotides +1 and -1, makes the nucleotide +2 point upwards
function of RNAPII “Fork” loops
positioned to open the DNA to form the transcription bubble
function of RNAPII “wall”
forces DNA to bend
function of RNAPII “rudder”
destabilizes RNA-DNA hybrid after ~1 turn to help RNA exit
function of RNAPII “lid”
maintains dissociation of the RNA-DNA hybrid
function of RNAPII “zipper”
promotes re-association of the DNA at the other end of the bubble
function of RNAPII “pore 1”
at the bottom of the “Funnel” serves to admit NTPs to the active site and to enable extrusion of nascent RNA if RNAP backtracks during proofreading
function of RNAPII “bridge”
“Bridge” helix plays a role in RNAP translocation: by alternating between a straight and a bent conformation it pushes the 3’ paired base of the RNA from the +1 to the -1 position
where does a-amanitin bind?
the “funnel” and makes the bridge helix not mobile, the polymerization of RNA stops, DNA template cannot move relevant to enzyme
CTD is what?
c-terminal extension of RNAPII
is a repeating 7AA sequence
can be differentially phosphorylated
preinition the CTD is what?
unphosphorylated (RNAPIIA)
what is RNAPIIO?
phosphorylated CTD
once RNAPII clears promoter what happens?
phosphorylasion of CTD so CTD can recruit RNA processing factors (splicing)
unphosphorylated CTD recuits what?
mediator complex and TBP (TATA box binding protein)
CTD with first ser5 phos recruits what?
histone modification/capping enzymes
CTD with second ser2 phos recruits what?
factors for transcript splicing
CTD with third ser2 phos recruits what?
3’ RNA end processing enzymes
