ethan hack material Flashcards
when end is RNA constructed from
5’
which end of an amino acid is positively charged and which is negatively charged
amine group (NH2) is positvely charged, carboxyl group (COOH) is negatively charged
name the 3 stop codons
UAG UAA UGA
what part of tRNA binds to the AA and the codon
anticodon binds to mRNA’s codon, 3’ end binds to AA
what type of 2D and 3D structure does tRNA have
cloverleaf (2D) and L shaped (3D)
what is the name of the enzyme that bonds AA’s to tRNA
aminoacyl-tRNA synthase
name a modified base that may be on the third position of a tRNA’s anticodon allowing it to bind to more than one codon
inosine
what is the location of translation
ribosomes
what are ribosomes made up of
protein and RNA
name the 3 binding sites on a ribosome and which sub unit are they one
Polypeptide and Anticodon, exit
small subunit
describe the initiation part of translation
1) first aminoacyl-tRNA binds to the P site of the small sub unit
2) codon-anticodon binding occours
3) large subunit binds
4) second aminoacyl-tRNA binds to the A site
discuss polycistronic DNA
multiple genes transcribed in a single mRNA molecule, like is done in prokaryotes from an operon
discuss prokaryotic RNA polymerase’s 2 forms
core enzyme; 5 subunits
holo enzyme; 6 subunits; also has a sigma factor
only the holo enzyme form can recognise the promotor region
how does prokaryotic RNA polymerase know where transcription stops
a ‘transcription termination sequence’ exists; mRNA folds into a stemp-loop structure which destabilises the mRNA-DNA interaction causing RNA to dissociate from the gene
how do prokaryotic and eukaryotic ribosomes know which codon is the first amino acid to match to an AA
prokaryotes; the shine-delgarno sequence of mRNA (close to start codon) binds to ribosomal RNA and acts as the ribosome binding site
eukaryotic; the ribosome binds to the begininning of mRNA and moves to the first AUG of mRNA
what is the shine- dalgarno sequence
AGGAGG. a sequence on prokaryotic mRNA which binds to the ribosome
discuss lactose structure
disaccaride consisting of 1 glucose and 1 galactose
discuss what the genes lacI, lacZ, lacY, lacA and lacI are expressed as and the function of the expressed products
lacA; beta-galactosidase transetylase; unknown function
lacI; repressor protein, binds to the operator region and stops transcription when lactose is absent
lacZ; beta-galactosidase; hydrolyses lactose
lacY; lactose permease; transports lactose into the e coli cell
discuss the 4 domains (polypeptides) of a a repressor protein
n terminal; binds to the DNA (operator region)
hinge region; holds N terminal in correct orientation
allolactose binding region
C terminal; used for two repressors to interact to form a dimer, which is needed for the repressor to bind to the DNA
discuss how allolactose interacts with repressor protein domain
allolactose binds to the allolactose binding region, and causes a conformational shape change which moves the hinge region; N terminal doesnt bind to DNA
why does binding of the repressor binding stop RNA polymerase action
operator and promotor sites overlap; binding of repressor protein means RNA polymerase cant bind
how many operator regions exist per operon. discuss these operators
3; one original operator and 2 auxillary operators. the auxillary operators are downstream of lacZ
binidng of repressor to 2 operators decreases transcription much more than binding of one repressor
what does i- mean
mutated lacI gene; mutated repressor protein which cant bind to the operon; transcription os constitutive (constant)
is the lacI gene part of the lac operon
no
discuss Oc (lap operon)
operator region is mutated; repressor cant bind; constitutive
discuss evidence for the lac operon mechanism
name the 3 scientists who worked on this
Jacob, Lwoff and Monod
looked at mutants with altered gene expression
the way that mutated beta-galactosidase (lacY) is screened for is by giving colonies the arteficial substrate X-gal, which is converted to blue product. those colonies which arent blue have a mutated gene and should be studied
when do lac operon mutation diagrams become complicated
when the cells are engineered to be diploid, cis and trans acting mutations now exist. trans mutation effect expression of genes in other DNA molecules to the one where mutation occoured
why is the lac promotor a weak promotor;
what is the name of a mutated promotor which isint weak, and why isint it weak
the -10 and -35 sequences are non standard
PlacUV5; -10 and -35 sequences are close to the consensus sequence
discuss of the importance of glucose concentrations being low in terms of lac operon regulation
when glucose concentration is low, cAMP concentrations are high; cAMP activates a CAP protein which binds to DNA’s -35 region, bends DNA 90 degrees. RNA polymerase binds to CAP with one of its sub units; increased binding to promoter occours if CAP is present
catabolic operons are switched (ON/OFF) when metabolite is present
Biosynthetic operons are switched (ON/OFF) when supply of product is low
ON
ON
discuss what the trp operon has genes for.
discuss how the operon’s expression is switched off
tryptophan AA
repressor only works when high concentrations of tryptophan exists, which binds to the repressor and allows it to bind to the DNA
define quorum sensing
discuss the bobtail squid being bioluminescent
discuss pathogenic bacteria
regulating gene expression in response to cell population density
symbiotic bacteria in the squids light organ are responsible for the light
these cells synthesise AHL (luxI gene), which diffuses out of the cell
if the cell density of bacteria is high enough then the concentration of AHL incrases, bacteria take it up, it binds to the LuxR gene, which activates transcription of the lux operon; light emmision
when invading a host, bacteria only produce degradative enzymes if the density of cells is high enough
discuss structure of a chromosome
DNA associated with histone proteins
telomeres (repetitive DNA) exist at the end of DNA, proteins attach to the telomeres
a nucleosome is a unit of DNA wrapped around a histone protein
why are DNA and histones bound so tightly
DNA is negatively charged, histones are positively charged
how many polypeptides does a histone protein consists of
8 (H2A, H2B, h3, H4 are all present twice)
discuss how many bp’s and genes are possessed by; yeast, plants, invertbebrates and vertebrates
bp;
12 mbp, 125mbp, 100 mbp, 3300 mbp
genes;
6000, 25000, 10000, 24000
define an intron and estimate how much of the human genome is makes up
a sequence of DNA that is transcribed but then removed from mRNA by splicing
20%
discuss how much of the human genome is made up of repetitive DNA
discuss simple sequence repeats and transposons
discuss the two types of transposons
50%
simple sequence repeats;
two nucleotides repeated
5% guman genome
helps organise chromosome structure
transposons; DNA that is duplicated and then moves to a new site
form via an RNA intermediate (require reverse transcriptase)
slow to move; often cause mutations leading to DNA repair or apoptosis
LINE’s; long interspaced elements (6000bp)
code for enzymes needed for transposition (reverse transcriptase)
20% human genome
SINE’s; short interspaced elements (200bp)
15% human genome; must be more frequent than LINE’s
Alu elements are a type of SINE, which rely on LINE elements for transposition
discuss % composition of human genome
introns 20% simple repeats 5% LINE's 20% SINE's 20% exons; 2%
discuss the 3 ways RNA is processed
discuss how intron removal occours
1) 7-methyl cap at 5’ end
2)cleavage of 3’ end and addition of Poly(A) tail
3) removal of introns via splicing
splicosome (made up of snRNP’s) perform this
a) splicosome binds to the 5’ end of intron and cuts it
b) lariat (loop) formed as the 5’ end is bonded to the 2’ of the other end of the intron
c)spliceosome binded to and cuts the 3’ end
d) exons are joined
define alternative splicing, discuss what % of human genome has this performed on them, and discuss an example of gene
a single gene can code for more than one protein if different exons are used; proteins usually have related but not identical function
60%
tropomyosin has 8 different mRNA’s
discuss pseudogenes. how many does the human genome have
genes that evolve by duplication but then mutate and lose their function
20,000
difference the principle of synteny
the idea that the order of genes within a chromosome is conserved over evolutionary time
comapre yeast and human genomes
yeast a more compact genome (more genes relative to genome size)
yeast have less repetitive DNA
yeast average gene size is much smaller
much of yeast genome is open reading frame (start to stop codon); little space between genes
yeast genes usually have no introns, and if they do have an intron they only have one
define euchromatin and heterochromatin and which type is accessable for transcription and which type of formation DNA is in for mitosis/meiosis
euchromatin (nucleosomes spread out and not tightly packed)
heterochromatin (nucleosomes are condensed and closely packed together)
euchromatin is required for transcription; the only packaging present is into nucleosomes
during cell division, DNA is in the heterochromatin form
discuss 2 methods of opening up chromatin for transcription
is ATP required
1) histone chemical modification; acetyl groups are added to the the lysin AA’s of the protein, causing the histone to lose its positive charge so the DNA-histone and histone-histone interaction decreases
2) chromatin remodelling is done by chromatin remodelling complexe enzyme (remodeler) to make DNA more accessible
a) move nucleosomes
b) remove histone protein
c) unwarap DNA from protein
ATP required
name the 3 types of eukaryotic RNA polymerase and what they transcribe
approx how many subunits do these enzymes have
RNA Pol 1; transcribes genes for rRNA (found in nucleolus)
RNA Pol 2; transcribes protein coding genes. is involved in RNA processing too
RNA Pol 3; transcribes genes for tRNA and snRNP’s
all have more than 10 sub units
discuss the name of the sequence where eukaryotic transcription begins,
discuss sequence length and how far away from from transcription starting point it is
TATA
8 bp long (TATAAAXX)
25 bp upstream of transcription start point
discuss the translation’s 3 stages
initiation
elongation
termination
how does ribosome know where to begin translation (there are lots of AUG codons in mRNA)
for prokaryotes, the ribosome binds to the Shine-Dalgarno sequence of mRNA (AGGAGG)
for eukaryotes, the ribosome binds to the beggininng of mRNA and moves to the first AUG in the mRNA
discuss the 2 elongation factors required for translation; their roles and their different names in prokaroytes/eukaryotes
EF-Tu (EF-1 in eukaryotes) carries aminoacyl-tRNA to the amino acid
EF-G (EF-2) binds to the A site of the small sub unit and moves the ribosome relative to the mRNA (resets it)
Both hydrolyse GTP for energy
discuss how eukaryotic transcription is initiated
over 50 polypeptides required activator proteins chromatin remodelling proteins TFIID (TBP); bend DNA 70 degress RNA POL 2 binds elongation factor p-TEFb phosphorylates the carboxy terminal domain of RNApol2 which allows transcription to resume after it has been paused
discuss what sequences are present in eukaryotes if no TATA box is present
BRE (30 upstream)
DPE (30 downstream)
TBP is still required
discuss genes with no TATA box and no other sequences to define where transcription starts
these genes code for housekeeping proteins; (needed in small amounts). transcription doesnt start at a specific base but dispersed initiation occours every 50bp along the sequence
define cis-regulatory element
a DNA sequence in a gene, which transcription factors bind to which regulates the rate of gene expression
10bp consensus sequence
what are the two mechanisms by which a transcription factor works
alters chromatin structure
interacts with the transcription complex to change how RNA pol 2 binds to the promotor
define core promotor, proximal promtor and enhancer
the core promotor is the region of DNA that RNA polymerase binds to
proximal promotors are the region next to the core promotor that contains Cis-regulatory elements
enhancer, cis regulatory element very far from the core promotor
discuss what the metallothionein gene is expressed as and the fucntion of that protein
discuss 2 of the response elements that increase expression as well as the associated TF
the metalliothionein gene is expressed as a metallothionnein proteiin which binds toxic damaging metals as well as metals that are needed, like Zn2+
when Zn2+ is present, it binds to an activator protein which then binds to the MRE response element
when glucocorticoid is present, it binds to the glucocorticoid receptior which is activator which binds to the GRE
where is the beta globin gene found and what is the TF for this gene
the beta globin gene is found in erthroid cells (RBC precursor) and is activated when the gata-1 TF binds
explain a mechanism by which steroid hormones regulate transcription
the steroid hormone diffuses into the cell, binds to an activator protein and changes how it interacts with the cis regulatory element
what is most important secondary messenger of peptide hormones
cAMP
what is the name of the group of TF’s which bind to steroid hormones
ligand responsive transcription factors (LRTF)
what type of steroid hormone glucocorticoids and what is one improtant glucocorticoid hormone
adrenal steroid (released by the adrenal gland)
cortisol
what two situations is cortisol released in what physiological effects does it have
(does it it inhibit or promote)
starvation/intensive exercise
blood sugar increase by activation of genes needed for gluconeogenesis (liver cells)
inhibit
when glucocorticoid is not present, what stops the protein it effects having impacts on transcription
Hsp90 chaperone protein holds the glucocorticoid receptor in the cytoplasm. cortisol binds to the protein and causes hsp90 to dissociate allowing the protein to enter the nucleus
why does removal of hsp90 chaperone allow for the cortisol recepetor cell to enter the nucleus
its nuclear localisation signal has been unmasked
what is the mechanism by which cortisol receptor alters transcription
the recpeptor remodels the chromatin which allows other TF’s to bind
name other groups of steroid hormones apart from adrenal steroids
thyroid hormones, steroid sex hormones, vitamin D, retinoic acid
why do certain cells not respond to hormones
they dont have the receptor
define somtie
a cluster of undifferentiated cells which form next to the notochord in vertebrate embryos
discuss the two steps of muscle development
somite neighbouring cells signal for somite cells to transcribe MyoD and Myof5 genes, which cause the somites to convert into myoblasts
myoblasts transcribe myogenin which causes the myoblasts to fuse and form a multinucleate muscle cell (myotubule)
define myoblast
undifferentiated cells which are precursors for muscle cells
whats the name of a mature muscle cell
myotubule
name the 3 genes needed for muscle development
MyoD and Myof5 (for somite to myoblast conversion) and myogenin (for myoblast to myotubule differentiation)
discuss evidence for the muscle development process
knocking out myoD and Myof5 means myoblasts dont form
knocking out Myogenin means myotubules done form
discuss how cells can be induced to become other cells
discuss 3 examples (cell that is induced, cell it can become, transcription factors, applications)
if different transcription factors are expressed, then different target genes are expressed so proteins for other cell types now exist
1) fibroblast>muscle cells myogening, Myof5, MyoD 2) fibroblast>iinduced pluripotent cells oct4, sox2, Klf4, Myo these cells can give rise to many cells
3)pancreatic exocrine cells>inset of Langerhans beta cells
Pdx1, Ngn3, MafA
cure diabetes
name two ways to alter gene expression apart from transcription
discuss an example of both
altering translating
mRNA for ferratin (iron storage protein) is only trnaslated when iron concentration is high
altering mRNA stability
trasferrin, (transfer protein used to take up iron when cell concentration is too low) mRNA is made more stable due to signalling for this low concentration
explain what a zinc finger protein is and an exmaple of one
proteins that consists of loops/helixes of amino acids. protein is stabilised by zinc ions
steroid hormone receptors (TF’s)
what the region of a zinc finger which binds to DNA
recognition helix
discuss the need for glucose as a regulator of the lac operon
the lac operon is weak due to non standard -10 and -35 regions in the promotor; glucose concentrations need to be low so that cAMP concentrations are high; cAMP binds to a catabolite activator protein (CAP) which binds to DNA’s -35 region, bends DNA and helps RNA polymerase bind
