genetics exam 3 Flashcards
what is the conservative model?
both parental strands stay together after DNA replication
2nd generation contains separate 15N and 14N
what is the semiconservative model?
the dsDNA contains one parental and one daughter strand after replication
2nd generation contains hybrid of 15N /14N and a separate 14N
what is the dispersive model?
parental and daughter DNA segments are interspersed in BOTH strands after replication
2nd generation only contains hybrid of 15N/14N
which DNA label is light and heavy?
15N = heavy media
14N = light media
how are nucleotides joined?
via phosphodiester linkages
which end has the hydroxyl and the phosphate?
5’ P
3’ OH
what direction does DNA polymerase catalyze polymerization?
5’-3’ (DNA template is 3’-5’)
what does DNA polymerase require to polymerize?
it cannot initiate DNA synthesis by linking two individual nucleotides together and can ONLY add onto PRE-EXISTING 3’ OH end of DNA or RNA
it requires a starting piece of RNA primer at the 3’ OH end (added by RNA polymerase)
what directions do the leading and lagging strands synthesize DNA in?
leading strand = DNA pol III attaches to the nucleotide in 5’-3’ and slides toward the OPENING of the replication fork
lagging strand moves AWAY from the replication, DNA pol III also attaches in 5’-3’ but contain okazaki fragments which will be sealed by DNA ligase
how many primers are needed in the leading and lagging strands?
leading = one primer
lagging = multiple primers
where does DNA synthesis begin in bacteria?
at site called oriC
each bacterial chromosome only has ONE origin of replication and proceeds BIDIRECTIONALLY
when does bacterial DNA synthesis end?
two replication forks will eventually meet at the opposite side of the chromosome, which ends the replication
what are the 3 types of DNA sequences in oriC that are functionally significant?
AT-rich region - where the two strands separate when the DnaA proteins bind to the DnaA boxes
DnaA boxes - DnaA proteins (help with bending of the chromosome) bind to DnaA boxes
GATC methylation sites - regulates replication , DNA adenine methyltransferase methylates A on both strands (immediately after replication, daughter strands are unmethylated, both the dsDNA is hemimethylated due to the parental strand)
INITIATION OF REPLICATION ONLY OCCURS EFFICIENTLY ON FULLY METHYLATED DNA
what is DnaB?
it is the bacterial chromosomal helicase that unwinds the dsDNA and replicates in 5’-3’ CW and CCW (bidirectional), using energy
bacterial replication contains TWO REPLICATION FORKS
what is the function of topoisomerase II?
aka DNA gyrase which travels ahead of helicase and alleviates the supercoils
what is the function of SS binding proteins?
bind to the separated DNA to keep them separated
what are the functions of DNA pol I and III?
involved in normal DNA replication
what is the function of DNA pol II, IV, and V?
needed for DNA repair and replication of damaged DNA
how does DNA pol III contribute to bacterial DNA replication?
it synthesizes the daughter strand
how does DNA pol I contribute to bacterial DNA replication?
removes RNA primers and replaces the RNA with DNA using 5’-3’ exonuclease activity to digest the RNA and a 5’-3’ polymerase to replace it with DNA
also has 3’-5’ exonuclease activity so that it can go backwards to correct its mistakes
how does DNA ligase contribute to bacterial DNA replication?
it seals the gaps in the sugar-phosphate backbone
what are the multiple proteins found at the replication fork called?
replisome complex
the 2 DNA pol II (on the leading and lagging strand) move as a unit during replication –> allows the coordination of leading and lagging strand synthesis
what is significant about lagging strand DNA synthesis?
the lagging strand is LOOPED –> allows DNA pol II to synthesize the okazaki fragments in 5’-3’ and move as a unit with the leading strand DNA pol III
completion of okazaki fragment –> enzyme releases lagging template strand, clamp loader complex reloads pol at next RNA primer and another loop is formed –> process repeats
what are the subunits of DNA pol III called?
DNA pol III holoenzyme
synthesizes DNA 5’-3’, 3’-5’ proofreading, clamp protein (allows DNA pol to slide along DNA without falling off - maintain association of DNA with pol II), clamp loader complex (helps clamp protein bind to DNA, uses ATP hydrolysis to open beta clamp and close it around template DNA)
what is on the opposite of oriC?
a pair of termination sequences aka TER SEQUENCES (T1 stops CCW and T2 stops CW)
Tus binds to ter sequences and stops the movement of the replication forks
only ONE ter sequence is required to stop one fork and the other fork ends its DNA synthesis when it reaches the stopped fork
what are the intertwined circular molecules at the end of bacterial DNA replication called?
catenanes which will be separated by topoisomerase II (used to prevent supercoils in dsDNA)
what type of chromosomes do eukaryotes have?
long, linear chromosomes
require multiple origins of replication
what is unique in the replication of eukaryotic chromosomes?
they form replication bubbles from multiple origins that merge into replicated chromosomes (begins and ends in S phase)
what is the origin of replication in S. cerevisiae called?
ARS elements
have high percentage of A and T
ARS consensus sequences (ATTTAT - A or G - TTA)
explain eukaryotic replication
begins with assembly of prereplication complex (preRC) which includes the origin recognition complex (ORC) which acts as the FIRST INITIATOR of eukaryotic DNA replication
other preRC proteins = MCM helicase, binding of MCM completes DNA replication licensing
activates in S PHASE, origins are ABLE TO BEGIN DNA SYNTHESIS
G1 phase = ORC binds to origin, other preRC assemble on origin, MCM binds to leading strand
S phase = preRC converted to active replication site by phosphorylation, MCM moves in 3’-5’ and DNA replication occurs bidirectionally
what are the function of the different eukaryotic DNA polymerases?
alpha, delta, and epsilon = nuclear DNA
gamma = mitochondrial DNA
alpha = polymerizing
beta = repair
what are the functions of delta and epsilon DNA pol?
delta = replication of LAGGING strand
epsilon = replication of LEADING strand
what are some differences between DNA replication in bacteria vs. eukaryotes?
bacteria = polymerases are also EXONUCLEASES, one origin of replication, Okazaki fragments 1000-2000
eukaryotes = not all polymerases are exonucleases, many origins of replication, Okazaki fragments 150-200, histones complexed to DNA
what is the function of DNA pol alpha?
associated with PRIMASE (10 RNA nucleotides followed by 20-30 DNA nucleotides)
exchange of DNA pol alpha for epsilon or delta is required for the elongation of leading and lagging strands = polymerase switch
what is the function of DNA pol in DNA repair?
DNA pol beta is NOT involved in DNA replication
plays role in removal of incorrect bases from damaged DNA
many are TRANSLESION-REPLICATING POLYMERASES (replication of damaged DNA, can synthesize a complementary strand over abnormal region)
what is the function of flap endonuclease?
removes RNA primers
polymerase delta runs into primer of adjacent okazaki fragment –> pushes portion of RNA primer into short flap and FLAP ENDONUCLEASE removes it
if flap is too long, it is cleaved by Dna2 nuclease/helicase (cuts long flap into short flap)
process continues until the entire RNA primer is removed and DNA ligase seals the two fragments together
what problem arises at eukaryotic chromosomal ends?
lagging strands CANNOT be replicated at 3’ end which can lead to chromosome shortening because unreplicated sequences at ends get shorter and shorter
also loss of important DNA sequences from ends of chromosomes –> problem resolved by adding telemores
what is the function of telomerase?
extends the ss PARENTAL template strand, lagging strand synthesis makes the complementary copy
contains protein and RNA
RNA is complementary to DNA sequence in telomeric repeat which allows telomerase to bind to 3’ overhang via RNA-DNA base pairing, rest of RNA acts as a template for addition of DNA to chromosome end
why are telomeres important?
linear eukaryotic chromosomes have telomeres at both ends
they are complex of telomeric DNA sequences and bound proteins which consist of moderately repetitive tandem arrays, ending in 3’ overhang (12-16 nt long)
telomeric sequences consist of several GUANINE nt and many THYMINE nt
what is the function of reverse transcriptase activity?
can copy RNA into DNA (normally you copy DNA into RNA to make mRNA)
describe the enzymatic steps of telomerase
- telomerase binds to the 3’ overhand
- telomerase synthesizes 6-nt repeat
- telomerase moves 6 nt to the right and begins making another repeat
steps are repeated many times to lengthen one strand of chromosome and other strand is extended by lagging strand synthesis using primer and DNA pol and ligase
what is the telomeric nt sequence in eukaryotes?
TTAGGG
what is TERT?
telomere reverse transcriptase
protein subunit of telomerase that is an RNA-dependent DNA polymerase
what cells express telomerase?
expressed only in embryo
tend to shorten in actively dividing cells
can shorten to 1,500 in elderly person
what is senescent?
cells become senescent when telomeres are short (stop dividing/growing)
insertion of highly active telomerase can block senescence
cancer cells carry mutations that increase activity of telomerase which prevents the telomere from shortening and senescence
what can mutations in genes involved in telomere function lead to?
genetic disorders like Werner syndrome or dyskeratosis congenita
what can the rate or telomere shortening tell you?
it is a good predictor of life span for different species (faster rate = shorter lifespan)
mean telomere length at birth is NOT a good indicator of lifespan
DNA base sequences define…
the beginning and end of a gene and regulate the level of RNA synthesis
control of gene expression occurs at?
initiation of transcription
what determines whether a gene will be transcribed?
regulatory proteins which are usually right next to the promotor (where RNA pol binds)
what is the function of regulator sequences?
DNA sites for the binding of regulatory proteins
role of regulatory proteins is to INFLUENCE RATE OF TRANSCRIPTION
what is the function of transcription factors?
proteins that recognize the promoter and regulatory sequences to control transcription
what does polycistronic mean?
bacterial mRNA may be polycistronic which means it encodes two or more polypeptides - a single mRNA may encode multiple proteins
the RNA transcript is complementary to…
the template strand
what are the stages of transcription in bacteria?
- initiation: promoter functions as a recognition site for RNA pol (RNAP), after binding –> DNA is denatured into a bubble known as open complex
- elongation/synthesis of the RNA transcript: RNA pol slides along the DNA in an open complex to synthesize RNA
- termination = terminator is reach that causes RNA pol and RNA transcript to dissociate from the DNA
what are examples of functional RNAs?
tRNA, rRNA
RNA components of:
- spliceosomes
- signal recognition particles
- telomerase (composed of RNA and proteins)
small regulatory RNAs
where are promoters located?
upstream of site where transcription of a gene begins
bases in a promoter sequence are numbered in relation to the transcription start site (+1)
what are consensus promoter sequences?
sequences in the -35 and -10 sequences in the promoters of many different genes that will be conserved if they are important
what does the RNA pol holoenzyme consist of?
core enzyme (makes the RNA, binds weakly to DNA and transcribes non-specifically) and a sigma factor (recognizes the -10 and -35 sequences)
altogether, holoenzyme can recognize promoters with specificity and transcribe DNA
how is transcription initiated in bacteria?
binding of RNA polymerase at promoter forms CLOSED COMPLEX (dsDNA)
OPEN COMPLEX is formed when TATAAT box in -10 region is unwound (ssDNA)
short RNA strand is made in the open complex, sigma factor is released at this point which marks the end of initiation
what happens to the DNA behind the open complex?
DNA rewinds back into a double helix
what is the rate of RNA synthesis?
43 nt per second
explain RNA synthesis
RNA pol slides along DNA, creating an open complex –> template DNA is used to make a complementary RNA-DNA hybrid –> RNA pol moves along template in 3’-5’ and RNA is synthesized in 5’-3’ (does NOT need primers)
TEMPLATE DNA STRAND AND mRNA ARE ANTIPARALLEL (mRNA is the same as the parental strand)
which ends are the promoters and terminators located?
promoters are always at the 3’ end
terminators are always at the 5’ end
what are the two different mechanisms that E. coli use for termination of transcription?
rho-dependent termination (REQUIRES protein known as r - rho) –> termination factor Rho catches up with RNAP at secondary structure and unwinds RNA from the hybrid in the transcription bubble, releasing RNA
rho-independent termination (DOES NOT require r) –> Rho-independent termination/intrinsic termination is facilitated by two sequences in the RNA
- URACIL RICH SEQUENCE located at 3’ end
- G-C RICH STEM-LOOP upstream of uracil rich sequence
how does eukaryotic RNA pol differ from bacterial RNA pol?
eukaryotes have 3 RNA pol while bacteria only have ONE
RNA pol I = transcribes all rRNA genes (except for 5S rRNA) –> these are transcribed as a single big RNA and then cut into pieces
RNA pol II = transcribes all PROTEIN-ENCODING gene (all mRNAs) and some snRNA genes needed for SPLICING
RNA pol III = transcribes all tRNA genes, 5S rRNA gene, microRNA genes
what are the eukaryotic promoter sequences?
they have a core promotor (important in determining precise start point for transcription) which consists of:
- TATA box (located in -25 sequence)
- transcriptional start site or initiator
and regulatory elements
CORE PROMOTER PRODUCES BASAL LEVEL OF TRANSCRIPTION
what are some factors that can affect eukaryotic transcription?
promoter-proximal elements
enhancers (stimulate transcription)
silencers (inhibit transcription)
both enhancers and silencers are often found in the -50 to -100 region
how does eukaryotic transcription differ from bacterial?
RNA pol II transcribes all pre-mRNAs
contains general transcription factors (GTFs) and a mediator (mediates interactions between RNA pol II and regulatory transcription that bind enhancers or silencers)
RNA pol (I, II,III) CANNOT bind directly to a promoter, there is NO sigma subunit –> instead GTFs first bind to the promoter and recruit RNA pol
(LECTURE 25, SLIDE 6)
explain the process of transcription in eukaryotes
TFIID binds to the TATA box (TFIID includes TATA-binding protein - TBP and what TBP associated factors - TAFs) –> TFIIB binds to TFIID, promoting the binding of RNA pol II to core promoter, TFIIF is bound to RNA pol II –> TFIIE and TFIIH bind to RNA pol II and form a preinitiation/closed complex –> TFIIH acts as a helicase to form an open complex and it also phosphorylates C-terminal domain of RNA pol II which helps release all the GTFs
in order for RNA pol II to leave, the c-terminal domain has to be PHOSPHORYLATED
how does transcriptional termination in eukaryotes differ from bacteria?
mRNAs in eukaryotes have POLY A TAIL added at 3’ end (RNA pol II transcribes the polyA signal into AAUAAA in RNA)
pre-mRNAs are then cleaved downstream of this signal, transcription terminates about 500-2000 nt downstream from polyA signal
what is colinearity?
sequence of DNA in non-template strand corresponds to sequence of nt in mRNA
sequence of codons in mRNA provides instructions for sequence of AA in polypep
do eukaryotic structural genes display colineraity?
not always
DNA sequences were found to be much larger than corresponding mRNAs because of R looping –> mRNA is interrupted by DNA loops which contain sequences only present in DNA and not mRNA
what does the pre-mRNA sequence consist of?
both introns and exons
introns will be later be removed via splicing and the exons will connect together to make mature mRNA
explain what is splicing in eukaryotic genes
initial transcript known as mRNA contains introns and exons which spliceosomes will splice before it can leave the nucleus
spliceosome is composed of snRNPS - each contains small nuclear RNA and a set of proteins
each subunit carries out different functions
- bind to intron seuqnece and precisely recognize the intron-exon boundaries
- hold the pre-mRNA in the correct configuration
- catalyze the chemical reactions that remove introns and covalently link exons
how does splicing occur?
intron contains seuqneces for splicing
- GU at 5’ end and AG at 3’ end
- internal BRANCHPOINT which contains A –> essential
U1 binds to the 5’ splice site (GU) and U2 binds to branch site (branchpoint A) –> U4/U6 and U5 trimer binds, looping intro out and exons are brought closer together –> 5’ splice site is cute, 5’ end of intron is connected to A, (FORMS A LARIAT or lasso) U1 and U4 are released –> 3’ splice site is cut, exon 1 is connected to exon 2, intron is released with U2, U5, and U6
INTRON IS DEGRADED
where does splicing occur?
in the NUCLEUS as RNA is being transcribed
what can some introns do?
they have the ability to self-splice
meaning intro removal in absence of snRNPs/factors, catalytic power is provided by RNA sequences in the intron
Group I and II introns
I = initiated by GTP
II = same steps as pre-mRNA with lariat intermediate (lasso)
what is alternative splicing?
pre-mRNAs may be spliced in many ways, creating different combos of exons that can be joined together to produce different mRNAs (can have exon skipping)
allows DIFFERENT polypeptides to be made from the SAME GENE (# of proteins produced in a cell may far exceed number of protein-coding genes in that cell)
how can RNA transcripts be modified?
final RNA differs from the initial transcript
mature mRNAs have 7-methyl-guanosine at their 5’ end aka CAPPING –> occurs as the pre-mRNA is being synthesized by RNA pol II aka co-transcriptional (cap-binding proteins recognize CAP structure on RNA, CAP is important for movement of some RNAs into the cytoplasm and INITIATION OF TRANSLATION OF mRNA)
mature mRNAs also have polyA tail which is NOT ENCODED in gene sequence, added after gene is completely transcribed, polyA tail is important for translation, export, and mRNA stability
what is the order of co-transcription of pre-mRNA?
initiation of transcription
addition of 5’ cap
splicing
addition of polyA tail
transport out of nucleus into cytoplasm
what is RNA processing?
cleavage of large RNA transcript into smaller pieces, one or more of the smaller pieces becomes a functional RNA molecule
- many nonstructural genes are initially transcribed as large RNA –> large RNA transcript is cleaved into smaller pieces
- transfer RNAs are also made as large precursors that will be cleaved at 5’ and 3’ ends to produce mature, functional tRNAs
what is the large rRNA transcript made by?
made by RNA pol I
large ribosomal subunit contains 5S, 5.8S and 28S
small ribosomal subunit contains 18S
explain processing of a precursor tRNA molecule
RNaseP (endonuclease) makes cuts in the tRNA sequence to remove unnecessary sequences and generate a mature molecule
RNaseD (exonuclease) removes nucleotides from the ends of the molecule
what is RNA editing?
change in the nt sequences of an RNA
can involve addition or deletion of bases
only a FEW SPECIFIC based in a FEW mRNAs are targeted for editing
ex: deamination converts RNA nt to new forms (cytosine to uracil, adenine to hypoxanthine)
another kind of editing uses small guide RNAs to add/remove bases
what are some examples of RNA editing?
trypanosome - primarily addition but sometimes deletion of one or more URACILS
slime mold - CYTOSINE additions
mainly cytosine to uracil and adenine to hypoxanthine
what are the arginine analogs?
ornithine and citrulline
explain genetic analysis in relation to arginine
if an arg- mutant grows on an intermediate, the block in pathway affected by the mutation is before the intermediate is made
ex: arg-2 can grow in citrulline so if you supply it with citrulline it can convert it to arginine but it CANNOT convert ornithine to citrulline
if a mutant grows on a particular compound but not on MM, the block is BEFORE synthesis of that compound –> the more compounds a mutant grows on, the earlier the pathway block is
the reason why Beadle and Tatum observed four different categories of mutants that could not grow on media without methionine is because
four different enzymes are involved in a pathway for methionine biosynthesis
what is the one gene-one enzyme hypothesis?
a single gene controlled the synthesis of a single enzyme
ex: normal strains = met was synthesized by cell enzymes but in mutant strains = genetic defect in one gene prevented the synthesis of one protein required in one step of the pathway to produce that AA
how can you determine the order in which compounds are produced?
earlier intermediates in the pathway = fewer number of mutants that will grow on it
later intermediates in the pathway = more mutants will grow on it
order of genes are decreasing in number
what types of frameshift will result in mutant vs. WT?
mutant = 1,2,4,5 deletions or additions
WT = 1 deletion and 1 addition (cancel each other out), 3 or 6 deletions or additions
how was the triplet code determined?
used phage T4 rLLB gene as an experiment
lots of +1 or -1 mutations available
these are readily combined by recombination
insertions or deletions of groups of 3 nt DON’T shift the reading frame
what are other characteristics of the genetic code?
it is NON-OVERLAPPING and continuous (commaless)
how was the genetic code determined?
determined through a series of steps
1. overlapping vs non-overlapping, punctuated vs. non-punctuated
2. # of letters in codon
3. # of nt per codon using T4 phage rII insertion/deletion mutations
4. # of nt per codon using synthetic mRNAs
5. ID OF INDIVIDUAL CODONS AND THE AA THEY ENCODE WAS ACHIEVED USING SYNTHETIC mRNAS and tRNA BINDING EXPERIMENTS
what are the stop codons?
UAG, UGA, UAA
peptide sequence is co-linear with mRNA sequence
these codons are recognized by release factors
more than one codon cannot specify the same amino acid (t/f)
false
what is unique about the genetic code?
it is universal
what are exceptions to the genetic code?
selenocysteine and pyrrolysine are sometimes called the 21st and 22nd AA
encoded by UGA and UAG
what factors are needed for translation?
mRNA, ribosomes, tRNAs, and protein factors
what are the components of the tRNA?
contains an acceptor arm (CCA 3’) that carries the AA for the codon
3 step loop structures = a D-arm (dihydroxyl uracil) and a T-arm (ribothymamide), variable loop, and anticodon
Gm is the wobble base
secondary structure = cloverleaf,
3D = it has an L-shape
what is the enzyme that attaches AA to tRNAs?
aminoacyl-tRNA synthetases (20 total, one for each AA)
they catalyze 2 step reactions involving 3 different molecules –> AA, tRNA, and ATP –> results in charged or aminoacylated tRNA
what is the wobble rule of tRNAs?
the genetic code is degenerate (multiple codons, or sets of three nucleotides, can code for the same amino acid during protein synthesis)
with exception of SER, ARG, and LEU (6 codons each), this degeneracy always occurs at the 3rd position
what is the wobble hypothesis?
a single tRNA can decode more than 1 codon because the first base of the anticodon (wobble position which pairs with the 3rd wobble base of the codon) can form additional non-W-C pairs
1st two positions pair according to AU/GC rule
G can pair with C and U
U can pair with G and A
wobble restricted to 3rd codon position (1st anticodon position)
1st and 2nd positive are strictly W-C pairings
what is significant about the wobble position?
wobble position of anticodon in tRNA is HEAVILY MODIFIED
I = inosine (base found in some tRNAs, derived from A) binds to U,C,A
AGG - UCU instead of UCC
AGU - UCG instead of UCA
UCG - AGU instead of AGC
where does translation occur?
on the ribosome
what are the functions of the subunits of the ribosome?
small subunit = decoding functions
large subunit = peptide bond formation functions
bacterial cells only have one type of ribosome in their cytoplasm, eukaryotic cells have two types of ribosomes - one in cytoplasm and others in mitochondria and chloroplasts
what are the 4 stages of translation?
- initiation (ribosomal subunits, mRNA, and initiator tRNA assemble together)
- elongation (ribosome slides along mRNA and synthesizes a polypep)
- termination (stop codon is reached and polypep is released from ribosome)
- recycling (recycle translational components, ribosomes split into subunits needed for initiation of new round of translation)
what are the 3 binding sties for tRNA on the ribosome?
A site (aminoacyl - enter)
P site (peptidyl where the amino acid is added to the polypeptide growing chain)
E site (where the tRNA is released)
anticodon of tRNA interacts with small subunit and the CCA 3’ end interacts with the large subunit
explain initiation of translation
always begins with binding of SMALL ribosomal subunit to mRNA (30S in bacteria, 40S in eukaryotes)
dedicated INITIATOR tRNA BINDS TO P SITE along with initiation factors
in bacteria, initiator tRNA = charged with formyl-met
in eukaryotes, initiator tRNA = only charged with met
initiator tRNA pairs with AUG
LARGE subunit binds to 70S (bacterial) or 80s (eukaryotic) ribosome
how do ribosomes know where to initiate in bacteria?
AUG is initiation codon that codes for Met at internal positions
in BACTERIA, there is a ribosome binding site upstream of AUG in mRNA = Shin-Dalgarno mRNA sequence is complementary to 3’ end of 16S rRNA in
mRNA-rRNA base pairing positions 30S over AUG
bacterial specific mechanism
how do ribosomes know where to initiate in eukaryotes?
using SCANNING mechanism
all eukaryotic mRNAs have CAP at 5’ end
eukaryotic initiation factors bind to 5’ CAP and recruit 40S small subunit
what are the steps of elongation?
tRNA binding/decoding in A site
peptide bond formation in P site
translocation and tRNA in E site leave s
how is the ribosome a ribozyme?
2/3 RNA and 1/3 protein
both of its principal functions - decoding and peptide bond formation - are RNA-based activities
it is an RNA molecule with a catalytic function
when can translation begin?
it can begin before transcription is completed (aka coupling) - DOES NOT OCCUR IN EUKARYOTES
bacteria lack nucleus so transcription and translation occur in the cytoplasm
in eukaryotes, transcription is in the nucleus and translation is in the cytosol
what are unregulated genes called?
constitutive genes = always on
they proteins that are continuously necessary for the survival of the organism
why is gene regulation important?
- metabolism
- response to environmental stress
- cell division
explain regulation of gene expression in transcription
Transcription attenuation is a regulatory mechanism that controls gene expression by causing transcription to end prematurely. It’s a common strategy in bacteria, and is used to maintain a desired level of gene expression and to respond to environmental signals
explain regulation of gene expression in translation
- translation repressor proteins - can prevent translation from starting
- riboswitches - can produce mRNA conformation that influences translation
- antisense RNA - can bind to mRNA and prevent translation from starting
regulation can occur POST TRANSLATIONALLY
what is the function of small effector molecules that increase transcription?
they affect transcription regulation
bind to regulatory proteins but NOT to DNA directly
may increase transcription
molecules are INDUCERS because they are INDUCIBLE
bind activators = cause binding to DNA
bind repressors = inhibit binding to DNA
what happens when there’s a repressor protein, inducer molecule, and inducible gene?
without inducer, repressor will bind to DNA and inhibit transcription
when inducer binds to repressor, DNA transcription can proceed
what happens when there’s an activator protein, inducer molecule, and inducible gene?
without inducer, activator protein will not bind to DNA and transcription will not take place
with inducer, activator will bind to DNA and transcription occurs
what is the function of small effector molecule that inhibit transcription?
COREPRESSORS bind to repressors and cause them to bind to DNA
inhibitors bind to activators and prevent them from binding to DNA and transcribing = REPRESSIBLE
what happens when there’s a repressor protein, corepressor molecule, and repressible gene?
without corepressor, repressor protein will not bind to DNA, transcription occurs
with corepressor, repressor protein will bind to DNA and inhibit transcription
what happens when there’s an activator protein, inhibitor molecule, and repressible gene?
without inhibitor molecule, activator protein can bind to DNA and transcription occurs
with inhibitor molecule, activator cannot bind to DNA, transcription does not occur
what kind of sites do regulators have?
DNA binding site and an allosteric site that binds small effector molecule
DNA binding site and allosteric site are coupled
what is enzyme adaptation?
a particular enzyme appears in the cell only after the cell has been exposed to the enzyme’s substrate
what DNA sequences does an operon contain?
promoter, operator, structural genes, terminator
what are the sequences of the lac operon?
LacI repressor = DNA binding protein which can block lacZYA, encoded on separate gene
Lac promoter = DNA site, where RNAP binds, initiates transcription
Lac operator = DNA site, where REPRESSOR binds, between promoter and Z
what are the structural genes of the lac operon?
lacZ = encodes beta-galactosidase (break down lactose into glucose and galactose, rearranges lactose into allolactose - inducer)
lacY = encodes lactose permease
lacA = encodes beta-galactoside transacetylase
what are the two in which the lac operon is regulated?
- repressor lacI responds to presence/absence of LACTOSE = inducible, negative control mechanism, inducer is allolactose, binds to lac repressor and inactivates it
- activator protein - responds to presence/absence of GLUCOSE
what does the mutation in lacZY result in?
lac- which is unable to use lactose
what does lacOc result in?
unable to bind the repressor so the lac operon is ALWAYS ON regardless if lactose is present or absent
it is CIS-ACTING (only affects expression of adjacent, downstream lac operon)
ex: WT lacZ cis to Oc = always on (in Oc/O+)
WT lacY in cis to O+, lacY on only when lactose is present
what does lacI result in?
lacI- is always on
lacI- is recessive to lacI+ (I+ is inducible)
one copy of lacI+ encodes enough protein to regulate 2 operons (I+ is TRANS ACTING, can regulate Z+ and Y+ on different DNAs)
what does lacIS result in?
it is a SUPER-REPRESSOR
represses even in the presence of inducer
lacIS is DOMINANT to lacI+
explain inducible and repressible regulation of operons involved in catabolism and anabolism
operons involved in catabolism are INDUCIBLE
operons involved in anabolism are REPRESSIBLE
what are translational regulatory proteins?
recognizes sequences within mRNA and regulate translation of mRNA
these proteins act to inhibit translation aka translation repressors
they inhibit translation in 2 ways:
1. binding next to the Shine-Dalgarno sequence and/or start codon (sterically hinder ribosome from initiating translation)
2. binding outside the Shine-Dalgarno/start codon region (stabilize an mRNA secondary structure that prevents initiation)
another way to regulator is via synthesis of antisense RNA
what is the funciton of proteim ompF?
important in osmoregulation
produced at LOW osmolarity, at HIGH osmolarity its synthesis is DECREASED
micF inhibits ompF at high osmolarity but does NOT code for a protein
micF is an antisense RNA (complementary) of ompF
what are some posttranslational regulation mechanisms?
feedback inhibition
phosphorylation
acetylation
methylation
what is the function of riboswitches?
it can regulate transcription of translation, or splicing
RNA exists in 2 different secondary conformations - one that allows gene expression and another that inhibits it
conversion between forms is due to binding of a small molecule, which is a product of the pathway being regulated
explain riboswitch of TPP
genes for TPP synthesis are found in the thi operon which is regulated by a riboswitch
riboswitch will determine if it continues or not (thiMD mRNA is ALWAYS made)
if TPP is low = transcription continues and mRNA is made
if TPP is high = terminator stem-loop forms and transcription is attenuated/terminated at U-rich sequence
what do the regulatory transcription factors influence?
influence ability of RNA pol II to being transcription of a PARTICULAR gene
what is combinatorial control?
every promoter’s activity is influenced by multiple transcription factors
common concepts contributing to combinatorial control
- one or more activator proteins may stimulate transcription
- one or more repressor proteins may inhibit transcription
- activators and repressors may be modulated by: binding of small effector molecules, protein-protein interactions, and covalent mods.
- regulatory proteins may alter nucleosomes near promoter
- DNA methylation may inhibit transcription by: preventing binding of activator protein and recruiting proteins that compact chromatin
what is important about enhancers/silencers?
they are orientation-independent
can function in forward or reverse orientation
most are located within a few hundred nt upstream of the promoter
how do regulatory transcription factors influence transcription by pol II in eukaryotes?
they DO NOT bind directly to RNA pol
repressors don’t block pol II binding and activators don’t recruit pol II directly
3 ways that communicate effects of regulatory transcription factors to the transcription machinery are:
- regulation via TFIID either directly or through coactivators/corepressors
- regulation via mediator
- regulation via alternations in chromatin conformation
what are the functions of coactivators?
proteins that increase transcription, but DO NOT BIND TO DNA DIRECTLY
what is the function of the mediator?
aid in phosphorylation of CTD of pol II, happens at the end of initiation, allowing it to enter elongation
phosphorylation allows pol II to move away from the promoter region and transcribe downstream DNA
repressor prevents phosphorylation, pol II is trapped at promoter and CANNOT proceed to elongation phase
when can the transcription factor bind to DNA?
it can only bind when a small molecule is bound to it
active form of TF is homo/hetero dimer
describe action of glucocorticoid hormones
hormone diffuses into cell, binds to a glucocorticoid receptor –> triggers release of Hsp90 –> NLS is now exposed –> 2 receptors dimerize and travel to nucleus –> GRE are DNA sequences that function as enhancers (located near dozens of different genes, so hormone can activate many genes
describe the activity of the CREB protein
cAMP acts as a second messenger that activates PKA –> phosphorylated CREB binds to DNA and stimulates transcription (CREB protein recognizes response element with consensus sequence 5’ TGACGTCA 3’) –> unphosphorylated CREB can bind to DNA but cannot activate transcription –> activation happens when CREB is bound by coactivator (CBP) which contacts the transcription machinery to activate it
what are the conformations of the chromatin?
closed = chromatin is VERY TIGHTLY packed, transcription may be difficult or impossible
open = chromatin is accessible to transcription factors, transcription takes place
what are the 3 types of changes to chromatin that affect accessibility of DNA?
- chromatin remodeling - moving nucleosomes on chromatin
- chemical modification of chromatin
- swapping common histones for variants
what are the 3 ways in which chromatin remodeling complexes change chromatin structure?
- change in position of nucleosomes (spacing to be more opened)
- eviction of histone octamers (histone removed leaving only DNA)
- change in composition of nucleosomes
what are the 5 histone genes?
H1, H2A, H2B, H3, H4
what is the histone code?
pattern of recognition mechanism
DNA transcription is largely reculated by post-translational mod to histone proteins
pattern of histone mods provide binding sites for proteins like TFs
these proteins bind based on histone code and affect transcription
what is acetylation associated with?
increased transcription of DNA
hypoacetylation = decreased transcription
acetylated lyse binds DNA less well, creates a more opened complex = increase transcription
why is the ChIP-sequence important?
it maps locations of specific nucleosomes within a genome
allows determination of:
- where nucleosomes are LOCATED
- where histone variants are FOUND
- where covalent mods of histones OCCUR
what is methylation associated with?
carried out by DNA methyltransferase
common in SOME EUKARYOTIC SPECIES, but not all
DNA methylation INHIBITS eukaryotic gene transcription
methylation of cytosine in CpG di-nt
ASSOCIATED WITH INACTIVE GENES (INHIBIT TRANSCRIPTION) and leads to gene silencing
methylation may influence binding of TFs
explain CpG islands
many genes contain CpG islands near their promoters
in housekeeping genes (expressed all the time) –> CpG islands are unmethylated, housekeeping genes are also expressed in most cell types
