unit 2 Flashcards
what did chase and hershey do?
demonstrated genetic material is DNA not Protein
using bacterophages (labelled DNA and protein and noticed that DNA was present in offspring and proteins weren’t)
what did chargoff do?
discover regularity in DNA base ratios by analyzing nucelotide composition
what are purines?
A and G
what are pyrimidines?
G and C
what is the equation for the relationship between purines and pyrimidines?
(A + G) / (T + C)
what did watson and crick do?
devised secondary structure of DNA
what was watson and crick’s discovery based on?
franklin and wilkins said DNA was a constant diameter helix
paulding created model building techniques
franklin said that phosphates were on the outside
what is the basic nucleotide strucutre?
phosphate group (attached to 5’ C)
base (attached to 1’ C)
deoxyribose sugar (OH at 3’ C)
what is the structural difference between purine and pyrimidines?
purines have two rings
pyrimidines have one rings
what is denaturing?
reversibly separating the strands
how is DNA denatured?
increased temp
reduced salt concentration
increased pH
solvents
what is Tm?
melting temp
how do you measure Tm?
absorbance goes up as duplex separates
what does Tm indicate?
duplex stability (higher higher)
what does Tm classify help with?
organism classification
detecting rare genetic mutations
molecular biological techniques (PCR, southern blotting)
how does DNA replicate?
semi conservative
how was the replication method of DNA proved?
meselson and stahl grew E coli and used equilibrium density gradient centrifugation to determine isotope composition of DNA
what is required for DNA synthesis?
template of ssDNA
all 4 dNTPs
DNA polymerase and other enzymes
free 3’ OH groups
which direction does DNA synthesis go?
5’ to 3’
what does DNA polymerase do?
uses dNTPs
catalyzes phosphodiester bonds
what are some limitations of DNA polymerase?
can only extend DNA
needs preexisting 3’ OH
only moves 5’ to 3’
where does DNA synthesis occur?
within the replication bubble in the replication fork (strands are synthesized simultaneously)
what is the DNA unit during synthesis?
replicon
what are the types of DNA replication for circular genomes?
theta replication
rolling circle replication
what is theta replciation?
entire replicon
bidirectional
one bubble and two forks
what is rolling circle replication?
no bubble
continuous
uncoupling of replicon
what are the steps of DNA replication?
initiation
unwinding
elongation
replacing DNA nucleotides
termination
what happens in initiation?
initiator protein binds to replicate origin
short DNA section unwinds and allows protein to bind
ss binding protein keeps them separate
helicase binds to lagging template and breaks H bonds
what happens during unwinding?
helicase breaks H bonds to separate strands
DNA gyrase travels ahead of fork to alleviate supercoiling by breaking and fixing the strands
what is DNA gyrase?
a topoisomerase that needs ATP
what happens during elongation?
Primase synthesizes rna primers
DNA poly 1 removes primers and fills w/ DNA nucleotides
DNA ligase smooths nicks in sugar/phosphate backbone
what happens during termination?
forks meet or reaches special sequence
how many DNA polys are used in eukaryotic replication?
alpha - initatites dna replication
delta - synthesizes lagging strand
epsilon - synthesises leading strand
how are origins activated in eukaryotes?
in clusteres when selected by relication licensing factors
what are telomeres?
ends of linear chromosomes (G-rch) that stabalize
what is telomerase?
reverse transcriptase that extends DNA and fills in gap
what is transcription?
selective, complimentary, antiparallel synthesis of RNA from DNA
how does transcription begin?
de novo (reads 3’ - 5’ and synthesizes 5’ to 3’)
what does transcription require?
DNA template
rNTPs
RNA poly and other proteins
what is the transcription unit?
region of DNA that codes for RNA
contains promoter, coding region, and termination site
what is the enzyme complex that completes transcription in proks?
holoenzyme
what are the steps of transcription?
initiation
elongation
termination
what are down and up mutations?
base subs that make sequence less similar (to reduce transcription rate) or opposite
what are the two terminators in bacteria?
rho-dependent
rho-independent
how does rho-dependent termination work?
rho binds to RNA upstream of the terminator
RNA poly pauses at term and rho catches up
rho unwinds DNA-RNA with helicase
how do rho-independent terminators work?
poly pauses at U’s and the hairpin turn destabilizes DNA-RNA and breaks a the chain of U’s
what is the promoter made up of in eukaryotes?
core promoter and regulatory promoter
what is the core promoter?
extends beyond start site and includes consensus sequence
what is the regulatory promoter?
located upstream and contains transcriptional activator proteins to bind to consensus sequence
how does termination work in eukaryotes?
doesnt require a specific sequence
cleabes mRNA at specific site
exonuclease degrades remaining mRNA afterwards
what is RNAi?
process where RNA inhibits gene expression
what are the two types of RNA in RNAi?
siRNA and miRNA
what are three ways RNAi does gene silencing?
condensing chromatin to suppress transctipion
inhibit transcription
destroy mRNA
where does miRNA come from?
primary miRNA that is encoded by genome
how is miRNA altered?
cleaved by Drosha (RNAase enzyme) into stem-loop
Dicer destroys stem-loop in cytosol
what is RISC?
complex with siRNA and miRNA and argonaute
what does siRNA do in RISC?
cleaves passenger strand, uses complimentary binding, cleaves mRNA
what does miRNA do in RISC?
discards passenger strand, uses incomplete complementary binding, cleaves and degrades mRNA, represses translation
how many targets do siRNA and miRNA have?
miRNA has multiple and siRNA has one
what is the pre-dicer form of siRNA?
dsRNA with less than 100 nucs
what is the pre-dicer form of miRNA?
pre-miRNA with 70-100 nucs with interspersed mismatches and hairpin structure
how do siRNA and miRNA cleave?
endonucleolytic
what are siRNA and miRNA complementary-ness?
siRNA is fully comp
miRNA is partially comp (usually 3’ UTR)
what is transcriptional gene silencing?
localized repressive chromatin formation (direct)
what is the colinear model of gene organization?
of nucs is proportionate to # of amino acids
what are the main parts of a gene?
DNA sequence, introns, UTRs
what are the main parts of mRNA in eukaryotes?
pre-mRNA has poly A tail and 5’ cap (and exons/introns)
mRNA has no introns (spliceosome)
is the colinear model of gene organization true?
yes of prokaryotes, not for uekaryotes
what are the main parts of mRNA in prokaryotes?
shine-Dalgarno sequence (right before start codon)
how do genes become proteins in euks?
DNA -> pre-mRNA -> mRNA -> (a single) protein
how do genes become proteins in proks?
protein-coding genes are usually in an operon (one start site for multiple genes) with no introns and no pre-mRNA
what are the types of pre-mRNA processing?
adding a 5’ cap
3’ cleavage and adding poly-A tails (polyadenylated)
RNA splicing (of introns)
what does adding the 5’ cap?
increased stability and helps with splicing
what is the process of adding a 5’ cap?
methylated guanine nucleotide is attached to 5’ end of pre-mRNA (5’ to 5’ linkage)
what does 3’ cleavage and poly-A tail do?
increased stability, helps get mRNA out of nucleus, helps ribisome-mRNA binding
what does RNA splicing require?
5’ splice cite, 3’ splice site, and a branch point
how does RNA splicing work?
introns are removed via lariat and exons are spliced together
what is the spliceosome made up of?
5 snRNAs
U1 and U2 base pair with consensus sequence at 5’ splice site and branch point site of pre-mRNA
what is the order of the pre-mRNA processes?
5’ cap
splicing
3’ poly A tail
what are the types of alternative splicing?
isoforms of proteins
exon skipping
intron retention
alternative splice sites
mutually exclusive exons
how can mutations affect splice sites?
disrupt consensus sequence
create new splice sites
initiate usage of cryptic sites
what is beta thalassemia?
genetic disorder caused by beta-globulin that causes anemia
what is RNA editing?
chemically altering indiviual nucleotides by enzymes
what are codons?
translational unit
can be start (AUG - met)
or stop (UAA, UAG, UGA)
what is tRNA?
have 5’ - CCA - 3’ sequence with anticodon arm and acceptor arm (where ends meet)
what is basic amino acid structure?
joined together peptide bonds
amino
carboxyl
side chain (r)
central carbon
are codons degenerate?
yes but not ambiguous
what types of codon degeneracy are there?
can be partial (changing last nuc MAY change aa)
or complete (changing last nuc wont change anything)
what is degeneracy supported by?
isoaccepting tRNA (bind one amino but recognise different codons)
wobble effect (one charged tRNA can pair with more than one codon)
where does the wobble effect happen?
3rd nucleotide of codon
what is isonine?
I nucleotide - intermediatry in tRNA that can pair with A, U, C
what are the types of mutations?
point mutations (nuc substitute)
frameshift mutations (insertion or deletion)
what does translation do?
converts genetic info into protein on ribosomes from 5’ to 3’
what are the types of point mutations?
missense (diff aa) nonsense (stop codon) or silent
what is required for translation?
mRNA template
tRNAs
amino acids
ribosomes
accessory proteins
energy from GTP hydrolysis
what are ribisomes?
RNA and protein complex
have two subunits
where does protein synthesis happen?
in the cavity between subunits in the ribisome
what are the sites on ribosomes for tRNA?
A (amino acyl) binding site
P (peptide) binding site
E (exit) site
what are the steps of translation?
tRNA charging
initiation
elongation
termination
what is tRNA charging?
attaches tRNA to the amino acid using energy at 3’ hydroxyl at the acceptor step to carboxyl on amino acid
what happens during translational initiation?
ribosomal subunits assemble at the translational start site
what does translation initiation require?
mRNA, subunits, initiator tRNA, initiation factors (IFs), and GTP
what happens during translation in prokaryotics?
IF3 binds to small subunits (large can’t bind), small binds to mRNA (base pair with shine-Dalgarno), IF factors leave and large subunit binds
what happens during translation elongation?
aa-tRNA enters A site -> peptide bond forms -> ribosome translocation -> tRNA exits from E site
what does elongation (translation) require?
aa-tRNAs, 70s initiation complex, elongation factors, GTP
what do EFs do in elongation (translation)?
guide incoming aa-tRNA to correct site
what does peptidyl transferase do in translation elongation?
catalyzes peptide bond formation
which direction does translation go?
5’ to 3’
what happens in translation termination?
ribosome translocates to a stop codon (no aa-tRNA enters A)
then release factors (RFs) bind to A and trigger release of polypeptide from P-site tRNA
what are operons?
group of bacterial structural genes under the control of one promoter (transcribed together)
what the parts of the operon?
PROG
promoter
regulator
operator
gene
what do regulator genes do?
help control expression of structural genes of the operon by increasing or decreasing their transcription
what the types of operons?
negative (inhibits)
positive (stimulates)
inducible (default = on)
repressible (default = off)
what is special about negative inducible?
synthesizes only when their substrate is available (economic)
what is special about negative repressible?
use the product to provide negative feedback
what is the lac operon?
negative inducible operon
who first described the lac operon?
jacob and monod
what is encoded by the lac operon?
b-galctosidase (lacZ - breaks down lactose)
permease (lacY - transports lactose into cell)
transacetylase (lacA)
what happens to lac operon in absence of lactose?
regulator (repressor) binds to operator and inhibits transcription
what happens to the lac operon in the presence of lactose?
converted into allolactose, which binds and activates regulator which allows transcription
what are the type of lac mutations?
structural
regulator
operator
promoter
what are strucutral mutations on the lac operon?
on lacZ or lacY
effect structure of proteins
independent
what are regulator gene mutations on the lac operon?
lacI
may create super repressor (no transcription ever)
what are operator mutations in the lac operon?
lacO
constitutive and cis-acting
produces beta-galactosidase with or without lactose
what are promoter mutations in the lac operon?
lacP-, interferes with binding RNA poly to promoter
don’t produce lac protein
if there is one normal gene it will work
what happens in E. coli with sugars?
prefers glucose over other sugars so does catabolite repression in the presence of glucose (turns off genes that process other sugars)
uses CAP (catabolite activator protein)
cAMP (produced when glucose is low)
what are the levels of eukaryotic gene regulation?
alteration of DNA or chromatin structure
transcriptional control
RNA processing and degradation
translational control
post-translational modification
what happens during the alteration of DNA or chromatin structure?
chromatin remodelling complexes bind to particular DNA sites to reposition nucs
histone modification and DNA methylation (to histone tails or DNA stops translation, adding acetyl groups to histones stimulates transcription)
what happens during transcriptional control of eukaryotes?
activator protein stimulate/stabalize basal transcription apparatus
enhancers (affect transcription of distant genes) and insulators (block enhancers like a wall)
what happens during RNA processing and degradation gene regulation?
alternative splicing generates different proteins
multiple 3’ cleavage sites produce different-length proteins
RNAi (inhibit translation)
what happens during translational control?
miRNAs inhibit translation of complementary mRNAs
availability of components affects rate of translation
when does gene regulation in proks happen?
mostly at the transcriptional level
is chromatin structure improtant?
yes for ukaryoties no for prokaryotes
