Duoma Flashcards
Central dogma of molecular biology
a constantly evolving theory of the transition of DNA to RNA to Protein via transcription then translation including the replication of DNA
Avery’s Experiment
found DNA is the genetic material
using bacterial transformation (a phenomenon where bacteria changes phenotypes depending on their environment, transformation is caused by the genetic material so if there is transformation occurring that means the genetic material is still present);
1. treated cell free extract with protease; mixed with R cell and still transformed therefore protein not genetic material
2. tested with RNAse and transformation still occurred
3. tested with DNAse and no transformation therefore DNA = genetic information
why do strands interact via hydrogen bonds and not covalent interactions?
covalent interactions would be too strong and wouldn’t allow for easy replication
how do phosphodiester bonds interact
covalently
OMP to 3’ OH antiparallel
Structure of DNA
2 unbranched polynucleotide chains running antiparallel interacting via hydrogen bonds
phosphodiester linkage connects each nucleotide within a chain
Chargaff’s Rule
the percentage of nucleic bases occur where A=T and C=G
varies between but not within species
stable over time
Watson-Crick Base Pairing
A binds to T via 2 hydrogen bonds while G binds to C via 3 hydrogen bonds
B-DNA
right handed helix with 10.5 base pairs per turn
base pairs lie flat and perpendicular to axis
propeller twist
exposed in major and minor groves
when is G-U base pairing allowed
when RNA base pairs with itself or another RNA molecule
Structure of RNA
linear single stranded polynucleotide chain with ribose sugar phosphate backbone (connected via phosphodiester bonds); A&U C&G,
base pair with complementary RNA or DNA antiparallel
genomes
complete set of genetic material present in a cell or organism
prokaryotes v eukaryotes
P: unicellular and lack nucleaus
E: DNA in nucleus
both go through DNA replication, transcription, translation
enzymes for processing genomes
Polymerases: synthesize polynucleotide chains
nucleases: digest polynucleotide chains
ligase: binding molecules together
replication
reproducing DNA from DNA
transcription
DNA to RNA, c before l
translation
RNA to proteins
rosalind franklin
used x-ray diffraction to study DNA structure
showed DNA is helical and has structural repeats that correspond to 3.4A and 34 A
photo 51 that shows double stranded and helical
phosphodiester linkage
OMP to 3’ OH
A-DNA
dehydrated DNA conformation
model of double stranded RNA and RNA DNA hybrid molecules
spores and sees
right handed helix
Z-DNA
seen in regions with high G-C content
left handed
denature
breaking hydrogen bonds between strands
anneal
forming hydrogen bonds between strands
gene
a unit of heredity; fixed in position
chromosome
DNA molecule that encodes genes
diploid
2 copies of each chromosome
haploid
one copy of each chromosome
DNA polymerase
adds one dNMP to the 3’ end of a growing DNA chain
require DNA template chain and primer
RNA polymerase
adds one NMP to 3’ end of RNA chain
require template chain
can initiate de novo synthesis of RNA
reverse transcriptase
adds one dNMP to 3’ end of growing DNA
requires RNA template chain and primer
endonuclease
breaks a phosphodiester bond within a polynucleotide chain
molecule specific, either DNase or RNase
excinuclease
breaks 2 phosphodiester bonds within a single polynucleotide chain
exonuclease
removes nucleotides from one end of a polynucleotide
either DNase or RNase
specific for digesting polynucleotide from either 5’ or 3’ end
exonuclease 1 is a 3’ to 5’ exonuclease
dna ligase
**only enzyme that links two existing DNA chains end to end by catalyzing formation of phosphodiester bond
template strand
what the polymerase reads in order to base pair; read 3’->5’ to make the new strand in 5’ to 3’ direction
what is the genetic material in both prokaryotes and eukaryotes
DNA
what are the repeating units of a DNA strand
deoxyribonucleoside 5’ mono-phosphates dNMP
what conformation of DNA is mostly used?
B-DNA
what yields complex secondary and tertiary structures?
internal base pairing within an RNA strand
what is a unit of heredity? what does it include?
a gene; includes DNA that encode something functional (RNA or protein) and the regulatory elements controlling expression
can the position of genes move
no, its fixed
where are nucleoside monophosphate added? how?
3’ end of developing chain via DNA or RNA polymerases
describe a nick
endonuclease cut only one strand
describe a double strand break
endonuclease cutting both strands
blunt break (same spot) or different (staggered cut)
what is special about palindromic sequence in DNA
read the same going forward as backward; a type of sequence that can be read for a sequence specific endonuclease
what are the types of endonucleases
sequence independent or
sequence specific/ restriction endonucleases/ enzymes
what does DNA ligase do?
put two existing chains of DNA together
how do phosphodiester bonds interact
covalently
OMP to 3’ OH antiparallel
what is special about palindromic sequence in DNA
read the same going forward as backward; a type of sequence that can be read for a sequence specific endonuclease
define replication and describe what occurs
when a cell duplicates its entire genomic DNA; each daughter cell inherits complete complement of the genetic information; occurs in S phase
describe the conservative method of replication
producing two DNA molecules one with both strands being old DNA and the other having completely new DNA strands
describe the semi-conservative method of replication
yields 2 DNA molecules, each with one daughter and one parent strand
describe the dispersive method of replication
yields two DNA molecules whose strands are hybrids of old and new genetic material; patchwork like
Describe the Meselson and Stahl Experiment
grew eccoli in food with only N15 and produced enough generations that the species only had N15 in it; took DNA and put through centrifuge (DNA on bottom because dense); put in N14 medium and allows one generation of reproduction; in the centrifuge, found a band in the middle; allows second generation in N14 medium, and 2 separate bands formed ; conclusion: semiconservative model because old strand is template to new strand being formed
how does the meselson and stahl experiment disprove the conservative method
disproving conservative model because of no evidence of old DNA left
how does the meselson and stahl experiment disprove the dispersive model
disprove dispersive since in the second generation there should be some only hybrid material, but a new only N14 strand exists
how does the meselson and stahl experiment prove the semiconservative model
old strand is template to new strand being formed as seen by the new band on top showing a DNA with complete new genetic material (when compared to the first generation before N14 was introduced)
what style of replication does DNA synthesis follow?
semi-conservative: one parent strand and one new strand
what catalyzes DNA synthesis
DNA polymerase; hand shaped; site in palm of your hand; goes in the 5’-> 3’ direction
what are the requirements for DNA synthesis
- DNA template string with a primer (RNA or DNA) with a 3’ OH
- substrates are the four dntp (adds dNMP and kicks of PPi)
- Mg2+ bc it stabilizes 3’ OH making a good nucleophile/ lowering pKa; stabilize alpha phosphate to make it a good electrophile where a phosphodiester bond forms and releases pyrophosphate; held by aspartic acid
describe the structural difference between deoxyribose and ribose
DR: H on C2 while ribose has OH on C2
compare and contrast DNA polymerase 1 and 3
1: roles in replication, recombination, and DNA repair; has 3’->5’ proofreading and 5’->3’ exonuclease activity ; low mutation rate
3: carries out genomic replication and DNA repair; 3’->5’ proofreading but not the other direction; high polymerization rate and processivity; low mutation rate
what is polymerization rate
how many nucleotides are added per second
what is processivity
how many nucleotides can be added before the enzyme falls off/ how stable it is
what mechanisms help avoid mistakes during replication
presynthetic error control
proofreading
mismatch repair
what is presynthetic error control? what enzymes perform it?
demands correct base pairing before phosphodiester bond forms due to filing in each other’s catalytic site; by all DNA Pols
what is proofreading? what enzymes are responsible for it?
only by DNA pol 1&3; remove mismatched bases in a backspace like fashion
what is mismatch repair? what enzymes perform it?
examine new DNA for mismatched bases after passing replication fork;
explain tautomers can mess up DNA replication checks
polymerases can be confused and accept a wrong base pair match due to tautomerization of bases to look like another’s catalytic site (ex. G & T); tautomers do not last long and dan pol 1 & 3 have mechanisms to prevent
what are the high-fidelity DNA polymerases? how are they special?
DNA Pol 1, DNA Pol 2 and DNA Pol 3; they have 2 active sites: one catalytic site for DNA synthesis and a 3’->5’ exonuclease site (proofreading)
what style of replication does DNA synthesis follow?
semi-conservative: one parent strand and one new strand; also bidirectional
define origin of replication
DNA sequence that starts synthesis (one for prokaryotes); proceeds bidirectionally until the DNA Pol’s hit each other; Polymerases stay at the replication fork
define replication fork
where parent DNA is being unwound
what does helicases do
unwind parent DNA
what replicates the separated strands of DNA
DNA polymerase
what is the leading strand
the strand being made in a continuous 5’ -> 3’ direction
what is the lagging strand
a strand that is made in small Okazaki fragments in the 5’ -> 3’ direction
how often does replication occur in a cell cycle?
once
what is the regulated step that controls DNA synthesis
initiation
describe DNA replication initiation in prokaryotes
beginning at the oriC (origin of replication), DnaA protein binds and recruits more proteins to create steric hinderance causing the DNA to pop at the DNA unwinding element (DUE)
describe DNA replication initiation in prokaryotes
beginning at the oriC (origin of replication), DnaA protein binds and recruits more proteins to create steric hinderance causing the DNA to pop at the DNA unwinding element (DUE)
what is the DNA unwinding element (DUE)?
an AT rich segment where strand separation occurs
what
describe DNA replication initiation in prokaryotes
beginning at the oriC (origin of replication), DnaA protein binds and recruits more proteins to create steric hinderance causing the DNA to pop at the DNA unwinding element (DUE); DnaC loads DnaB helicase on the each side of the oriC on the soon to be lagging strand; DnaC falls off, DNA Pol 3 gets on, DnaA comes off
describe DNA replication initiation in prokaryotes
beginning at the oriC (origin of replication), DnaA protein binds and recruits more proteins to create steric hinderance causing the DNA to pop at the DNA unwinding element (DUE); DnaC loads DnaB helices
what part of initiation commits a cell to replicating?
loading DnaB helicase
what are the parts to the DNA pol 3 holoenzyme?
core enzyme plus the “accessory” clamp loader and beta clamp
what is the function of the core enzyme of the DNA Pol 3?
catalyzes DNA synthesis
what is the function of the beta clamps of the DNA Pol 3?
tethers core enzyme to DNA, increases processivity
what is the function of the clamp loader of the DNA Pol 3?
scaffold for DNA Pol 3, assembles beta clamp onto DNA via ATP; coordinates with replication fork by interacting with DnaB helicase via tau subunits
what is primase?
an RNA polymerase that does not require a primer but does only bind to DNA template strands
what is the function of single stranded binding proteins
binds to DNA and protects it from secondary structures binding to it; keeps the DNA strands apart
Describe DNA elongation for the leading strand
primase primes once, and one core polymerase generates a continuous production of nucleotides
describe DNA elongation for the lagging strand
primase adds primer as the fragments are made, new beta clamp loaded at each RNA primer by clamp loader, 2 core enzymes are working here, go until they reach the previous primer where core polymerase pauses and releases beta clamp and transferred to newly loaded beta clamp; old clamp left behind (helps cleaning)
how is a lagging strand finished
remove primers with DNA Pol 1 (using 5’->3’ exonuclease) and fills gap with DNA; DNA ligase repairs nick using NAD+
describe what the DNA looks like after elongation in prokaryotes
DNA strands end in a “catenated” or looped state
what are positive supercoils
over winding of DNA helix (CW)
what are negative supercoils
underfunding of DNA helix, increased distance, (CCW)
what causes supercoils
a lot of torsional strain
describe the role of topoisomerases
relieve supercoils by breaking and rejoining strands
what is a topoisomerase type 1
single stand break, pass the intact strand through the break and seal the nick, no ATP
what is a topoisomerase type 2
double strand break, pass an intact segment through break and rejoin, uses 2 ATP
what decatenates the DNA
topoisomerase 4 ( a type 2 topoisomerase) separates the DNA strands
describe the differences between eukaryotes and prokaryotes of DNA replication
- multiple origins of replication in eukaryotes
- licensing for replication initiation
describe licensing
ensures DNA replication only occurs once in a cell cycle: when origin of replication complexes are bound tightly to DNA in early g1; CDC6 joins followed by Cdt1 and MCM (helicase)
what initiates DNA replication in eukaryotes
phosphorylation of proteins by cdk and DDK; tart of s phase, stops helicase recruitment; assembles replisome
what is a replisome
the combination of the proteins that carry out DNA replication
what is the core polymerase equivalent in eukaryotes for the leading strand
DNA pol epsilon
what is the core polymerase equivalent in eukaryotes for the lagging strand
DNA Pol delta
what is the priming equivalent in eukaryotes
DNA Pol alpha-primase
what is the helicase in eukaryotes
MCM
what is the clamp loader in eukaryotes
RFC
what is the clamp in eukaryotes
PCNA
what is the protection proteins (SSB equivalent) in eukaryotes
RPA
what is the ligase equivalent in eukaryotes
still DNA ligase
what is the DNase equivalent in eukaryotes
FEN1- clips off overhang
What catalyzes transcription
RNA polymerase
what is transcription
the process of DNA template-dependent RNA synthesis
messenger RNA (mRNA) function
houses the sequence of bases that encode for primary amino acid sequence for a protein; template of translation
transfer RNA (tRNA)
carries amino acid to catalytic site of ribosome; base pairs with mRNA to ensure the right amino acid was delivered
ribosomal RNA (rRNA)
structural component of a ribosome
template strand
the strand of DNA that transcription is based off of, reverse complement of coding strand and RNA primary transcript
coding strand
the strand not involved in transcription; DNA equivalent of the RNA transcript
RNA primary transcript
the new strand being formed as a reverse complement of the template; resembles the coding strand; finished RNA molecule
what are trans-acting factors
a substance that activates cis=acting elements for activation of transcription; diffusible; typically DNA binding proteins
what are cis-acting elements
a DNA sequence that promotes transcription
where is the transcription start site
+1
promoter
where transcription starts; cis-acting element in genome where RNA polymerase binds to initiate transcription; in prokaryotes, exist at the -35 region, -10 region with a speaker between those areas on the CODING strand
terminator
where transcription ends
gene
the DNA encoding a protein and it regulatory elements
what is special about the primary transcript product in bacteria
it is not modified and is used as mRNA for translation
open reading frame (ORF)
sequence of bases that encodes the primary sequence of a protein; coding sequence; from promoter to terminator
operons
coordinately regulated gene clusters
polycistronic mRNA
multiple ORFs encoding a different protein but one promoter and terminator in a bacterial cell
consensus promoter
a typical sequence seen to be a promoter; closer to the consensus sequence, the more likely to start transcription; TTGACA-N16-18-TATAAT-N5-9CAT; on CODING strand
what is the major determinant of gene expression in bacteria?
rate of transcription initiation - influenced by similarity to consensus sequence
constitutive promoter
what promoter sequence exists on the coding sequence; strength related to similarity to promoter consensus sequence
describe the RNA polymerase holoenzyme
subunit sigma + core enzyme (subunit alpha2 , subunit beta, subunit beta prime, subunit omega)
what is the role of the subunit sigma
recognizes a promoter
what does the subunit alpha2
essential for enzyme assembly and interact with activators
subunit beta and beta prime role
form the catalytic core
subunits omega role
provides structural stability
what is required for RNA polymerase
DNA template chain; no primer, can synthesize RNA de novo
describe RNA polymerase’s role in transcription
forms phosphodiester bond between 2 NTPs to being synthesis, 5’ end has 3 phosphate groups; no exonuclease activity (high error rate)
describe transcription initiation
sigma 70 identifies promoter region, holoenzyme binds to promoter to form closed complex; 12-15 bp unwind and form transcription/ ope complex; synthesizes 10 nt 1 nt/sec
describe transcription elongation
dissociation of sigma factor allows RNA polymerase to complete promoter clearance; elongation at 50-90 nt/sec,
describe other processes that occur during elongation
formation of RNA secondary structures slow down formation; topoisomerases relieve supercoiling
describe transcription termination
release of RNA and dissociation core enzyme from DNA; Rho independent or dependent which is determined by what is encoded in the genes
describe rho independent termination
no Rho protein needed, termination signal in RNA chain sequence - formation of stale hairpin structure followed by a bunch of U’s causing dissociation
describe Rho dependent
requires rho protein; rho protein is a helicase that binds at rut site and travels along new RNA being made using ATP; transcription terminates when rho and RNA polymerase (who will slow down due to secondary structures) touch
constitutive promoters
always available
inducible promoters
promoters that are active in certain environments and not in others; environment detected by transcription factors
transcription factors
regulates gene expression; trans acting facts that combine with cis acting factors in DNA to regulate transcription; interacts in sequence specific manner; read DNA by acting in minor or major grove where exposed
recognition helix
the alpha helix in the DNA binding protein that participates in hydrogen bonds and van der waals interactions with base pairs; determines where a transcription factor should bind to promote transcription
what is the main DNA binding domain motif for prokaryotes
helix turn helix; results in induced bend
what is the difference between reading the major groove versus the minor groove
while transcription factors can distinguish between base pairs and the order in which they are presented due to more functional groups being present, minor grooves can only recognize which base pair is present, not the order; this is because a major groove has more sequence specificity than a mini, explaining while there is more recognition on the major
dimeric DNA binding proteins
proteins with 2 subunits; if homo-, 2 of the same subunit; each part has own recognition helix; since both are looking for same sequence (palindromic sequence), increases SPECIFICITY and STABILITY
repressors
transcription factors that decrease the rate of transcription from a promoter; negative regulation; ligand control activity; regulate inducible promoters
activators
transcription factors that increase the rate of transcription from a promoter; positive regulation; ligand controlled, regulate inducible promoters
what is the importance of location of operator a repressor binds to
if on promoter, RNA polymerase cannot bind; if downstream, inhibits promoter clearance
where do activators bind
activator binds to a positive regulatory element located UPSTREAM of promoter, recruits RNA polymerase to weak promoter
describe the funtion of the lac operon
regulates lactose metabolism, glucose consumed first though so in low glucose high lactose environments, there is an up regulation of the lac operon
describe the regulation of the lac operon
glucose and cAMP are inversely related, allolactose and lactose are directly related; negative regulation occurs at lacI gene upstream and encodes lac repressor in absence of allolactose; positive regulation involves CRP binding DNA in presence of cAMP (low glucose), also upstream
describe regulation of lac operon in terms of glucose and lactose levels
high glucose (low cAMP ) low lactose: basal transcription
high glucose (low cAMP) high lactose: low transcription (use up glucose)
low glucose (high cAMP) high lactose: high transcription
low glucose (high cAMP) low lactose: basal transcription (activated but repressed)
small nuclear RNA (snRNA)
small RNA molecules that guide post transcriptional base modifications in tRNA, rRNA, and snRNA
microRNA (miRNA) and small interfering (siRNA)
act on mature mRNA to decrease translation
how are eukaryotic genes organized for transcription?
they stand alone in single transcription units, each having its own promoter and terminator
intron
sequences that will be eliminated during RNA processing (non coding region)
exon
coded region ; included in mature mRNA
primary transcript
the initial product of RNA including introns and eons
how are eukaryotic cells specialized
gene expression regulation defines properties of cells in order for there to be different cell types
chromosome
contains DNA molecule that encodes genes
chromatin
proteins bound to chromosomal DNA
euchromatin
light staining material; open chromatin structure where genes are available for transcription
heterochromatin
dark staining matter; condensed, silenced genes
constitutive heterochromatin
always condensed state; typically areas with no genes
facultative heterochromatin
revert to euchromatin in response to cellular cues; differers between cell types
nucleosome
basic unit of chromatin; contain histone core and around 200 basepairs
what is interesting about histone core contacts
they are sequence independent; electrostatic interaction and H bonds occur between (+) histone and (-) sugar phosphate backbone
what is the structure of the histone core
2 copies of each: H2A, H2B, H3, and H4,
1 histone H1 to lock DNA to nucleosome
what is chromatin structure produced
as a product of combined actions of epigenetic mark (histone modifications, DNA methylation, trans acting transcription factors)
“pioneering” transcription factors
only thing that can access condensed chromatin
what does epigenetic mean in terms of transcripition
above genetics; mediators of epigenetic are COVALENT modification of the chromatin or DNA that do NOT affect the DNA sequence; not chaning DNA sequence, just how it is being used
what is the function of histone tails and its role in transcription regulation
the N termini of histones H3 and H4 and both termini of N and C of H2A and H2B; posttranslational modifications regulate chromatin structure
what is associated with closed chromatin
hypermehtylation of histone tail; HDAC and HMT
what is associated with open chromatin
histone acetylation of histone tail; HAT
histone acetyltransferase (HAT)
acetylates lysine in histone tails
histone deacetylase (HDAC)
removes acetyl groups from histone tails
histone methyltransferase (HMT)
methylates lysine and arginine in the histone tails
compare methylation versus acetylation
acetylation has both HAT and HDAC making it readily reversible, methylation is harder to reverse making it more stable
describe how to read the histone code
- denote which histone protein you are talking about
- denote which amino acid you are talking about with its one letter code and position
- tell whether it was acetylation or methylation with the number
what does the code H3K4me2 mean
H3 protein was methylated twice at the lysine-4 position
how does acetylation and methylation interact with the nucleosome
Ac: neutralizes positive charge on lysine, reduces electrostatic attraction (opening chromatin structure), factors recruitment of chromatin remodeling complex; associated with transcriptional activation
Me: stabilizes positive charge on lysine/ arginine; effect on transcript depends on amino acid position and # methyl groups (hypo- helps in transcription while hyper- no )
chromatin remodeling complex
remodels chromatin structure via unwrapping DNA from nucleosome, repositioning nucleosomes, or evicting nucleosomes ex. SWI/SNF
what is an example of an epigenetic modification than can occur to the DNA (not just RNA transcripts)
hypermethylation of CpG island near a promoter silences that promoter
CpG = cytosine that can be methylated by DNA methyltransferase (DNMT)
doesn’t change base pairing; impacts how transcription factors bind since it is sticking out into grooves - silencer
describe eukaryotic transcription initiation
starting with condensed heterochromatin state, pioneering transcription factors bind with 2 dominos (one binding to DNA and the other (activation domain) to recruit enzymes via protein protein interactions to promote opening, also recruiting chromatin remodeling complex (order does not matter)
HAT, pioneering transcription factor, and chromatin remodeling complex
ex. recruit HAT
SWI/SNF
chromatin remodeling complex, binds to pioneering transcription factor or HAT; exposes DNA and promoter for transcritpion elongation to begin
RNA polymerase in eukaryotic cells
RNA polymerase 2: makes mRNA and some small RNAs
RNA polymerase 1: synthesizes ribosomal rRNA
RNA polymerase 3: generates tRNA and other small RNAs
what is the difference between eukaryotic and prokaryotic RNA polymerase
many more subunits in eukaryotic cells/ bigger complex than prokaryotes; 3 types; no sigma unit in eukaryotic; cannot bind to DNA on our own, must recruit transcription factors that can recruit RNA polymers
what is unique to RNA pol 2
c-terminal domain (CTD): important in transcription initiation and post transcriptional modification
what is specific to mammalian genes
they have promoter proximal elements but also enhancers at distant locations along the chromosome; binding sites for transcription factors
promoter proximal elements position
-40 to -200
enhancers location
promoter distal positions, house cluster of binding sites for transcription factors; relies on DNA bending from HMG proteins to get closer to the promoter area
what are some eukaryotic sequence specific DNA binding motifs
helix turn helix - also in prokaryotes
homeodomain - important in prenatal development
classical zinc finger
nuclear receptor finger
leucine zipper proteins
helix-loop-helix proteins
classical zinc finger
zinc ion in the middle between 2 six and 2 his, 2 antiparallel beta strands and a alpha helix, conserved phe/ tyr and leu for structure
describe what occurs when there a multiple zn finger in one protein
each motif can have the same or different recognition helixes, but each helix makes its own sequence specific bonds to increase stability and specificity
nuclear receptor zinc finger
bind as a dimer, each with own recognition helix and second helix for structure, four cis on Zn,
leucine zipper proteins
have series of leucine aligned along alpha helix that participate in protein protein interactions for dimerization; recognitionhelices = extension of leucine containing helices
helix loop helix
dimerization domain; extension of alpha helix is recognition part
eukaryotic activators
exert positive gene regulation; modular design with sequence specific DBD, flexible hinge, and activation domain
what can activation domains interact with
coactivators, HAT, chromatin remodeling comlex, mediator, preinitiation complex
the difference between an activaor and a coactivator
a: binds DNA in sequence specific manner;
c: does not bind to DNA
describe an example of how activators work in terms of the the glucocorticoid receptor
a nuclear receptor, binds a ligand to be an active transcription factor that will then find its specific cis acting element; activates genes that recruit coactivator that binds to activation domain; coasviator with HAT activity, mediator, and indirectly with the preinitiation complex and RNA pol 2
stimulation transcription to get RNA pol to promoter
high mobility group (HMG) proteins
when binds to DNA can create extreme bends
mediator
does not bind DNA, a protein that binds to activators/ coactivators/ preintiation complex and creates a bridge between them; all of this to recruit RNA pol 2 to promoter
transcription preinitiation compleX
TFIID, TFIIA< TFIIB, TFIIF/ RNA POL 2, TFIIE, TFIIH,
transcription preinitiation complex process
TFIID binds at the promoter.
* TFIIA may join the complex.
* TFIIB binds to DNA and TBP. - creates docking site for RNA pol 2
* TFIIF/ RNA pol II join the complex by binding to TFIIB.
* TFIIE and TFIIH enter the complex in succession.
* TFIIH is a complex with two distinct functions whose completion finalizes preparation for transcription to begin
- Mediator must be present and interacting with CTD to get RNA Pol 2 to promoter
what are the two function of TFIIH
DNA helicase to generate the transcription bubble.
Protein kinase that phosphorylates the RNA pol II CTD to initiate transcription. - last step in prepping for transcription
what gives the “green light” for transcription
all activators are bound to promoter proximal, enhancer, regions, coactivators, HATs and interaction with mediator and preinitiation complex
not just the presence of TFIIH
TAT binding protein (TBP)
locates and binds to TATA boxes in eukaryotic promoters, minor groove, makes up TFIID
TBP-associated factors (TAFs)
function in correlation with the recognition of other sequence elements, make up TFIID
how do eukaryotes express negative gene regulation
repressors are transcription factors that inhibit transcription by
1. competitive binding (displace activator)
2. binding to activator to prevent interaction with mediator (corepressor)
3. altering preinitiaion complex assembly
4. provide docking for HDAC
how can eukaryotic transcription be repressed by chromatin modification
- repressor with DBD that binds to a negative cis regulatory element
- repression domain (RD) recruits HDAC to remove acetyl groups (which condenses chromatin)
describe how methylation can silence genes
ex. H3K9
HMT methylates H3K9 which provides a docking site for heterochromatin protein (HP1) which recruits more HP1
interactions here compact the chromatin
combinatorial control
coordinate action of transcription factors give rise to different regulatory factors causing each cell type to be able to have a different population of transcription factors that tell it which cell type it is going to be n
what occurs in eukaryotes than doesn’t in prokaryotes?
posttranscriptiona RNA processing to produce a mature mRNA with a 5’ cap and a 3’ poly-A-tail
why is phosphorylation of the C terminal Domain of RNA pol 2 nexessary
- promoter escape
- docking site for capping enzyme, cap binding enzyme, cleavage/ termination complexes and RNA splicing factors
describe the assembly of the 5’ cap
done by the capping enzyme to provide protection from the 5’->3’ exonucleases and for translation initiation
cap = methylated guanine (via SAM) and 3 P’s with multiple catalytic sites:
1. phophohydrolase: remove gamma phosphate
2. guanylyl transferase: add guanine nucleotide (GMP) via GTP and release PPi
occurs when 5’ end comes out of transcrip
describe transcription termination
RNA pol 2 continues transcription beyond the termination sequence; termination factors bound to CTD recognize cleavage sequence and bind to RNA; termination when cleavage of endonuclease and RNA pol2 dissociates
describe the assembly of the poly-A-tail
undergoes polyadenylation by polyadenylate polymerase or poly-A polymerase on the 3’ OH
doesnt require template
describe RNA splicing
removal of intron sequences and link exon sequences; fixed order of exons but not expression
describe splice sites
5’ splice site at the upstream 5’ end of a intron with GU
3’ splice site at the downstream end of a 3’ intron with AG
GU/ AG rule
branch point A at some point in middle and a pyrimidine rich region between branch point and 3’ splice site
what is/ makes up the spliceosome
the nuclear complex responsible for removing introns and bounding exons
made up of majority snRNP (small nuclear ribonuclear protein) which is made up of snRNA
U1-U6 subunits
what is the function of U1 snRNP
binds to 5’ splice site in RNA by recognizing GU base pair ; U1 has pseudouridine that can bind to many different nucleotides
what is the function of U2 snRNP
binds to branch site and aligns it for the 1st splicing reaction; binds with sequences around A but not A itself causing it to bulge
what is the function of U4 snRNP
binds to and holds U6 snRNP so it doesn’t chop randomly
what is the function of U5 snRNP
aligns pre-RNA for 2nd splicing reaction
what is the function of U6 snRNP
promotes catalysis of splicing reactions; held by U4
describe the sequence of events of the assembly of the spliceosome
- U1 binds to 5’ base site
- U2 binds around branching point
- U5 and U4/U6 bind to complex to complete assembly
describe, after assembly, how the 1st splice activity occurs
U2, U5, and U6 base pair with each other to align the branch site with the 5’ splice site; connecting introns, produce lariat structure with 2’ to 5’ phosphodiester bond
2’ OH to 5’P of intron
describe the 2nd splice activity done by the spliceosome
U2, U5, U6 rearrange to bring 5’ and 3’ sites together, connecting exons, form splice junction
3’ OH to 5’P of exon
describe alternative splicing
mechanism for developmental or tissue specific production of differing mRNAs; programmed inclusion or exclusion of EXONS in different cell types
does NOT change the order, only the exons that are expressed
describe the regulation of selecting exon sequences
sequence-specific RNA binding proteins
splicing activators: SR proteins promote splicing by binding to exonic splicing enhancers (ESE)
splicing repressor: heterogeneous nuclear ribonuclearproteins (hnRNP) bind to exonic splicing silencers (ESS) and inhibit splicing
describe alternative exon / exon skipping
complete bypass of an exon
describe alternative 5’ splice sites
a different 5’ splice site; not skipping an exon just splicing earlier/ later than another one
describe alternative 3’ splice sites
a different 3’ splice site; not skipping an exon just splicing earlier/ later
describe mutually exclusive alternative exon
both expressions exhibit exon skipping but of different exons
describe alternative promoter and first exon
start at completely different points
describe alternative poly(A) site and terminal exon
end at different points
what occurs after splicing is done
mature RNA exits the nucleus through a pore in order to go into the cytoplasm for translation
codon
3 bases that specifies one amino acid
degenerate code means what
most amino acids are encoded by more than one codon
what are the stop codons
UAA UAG and UGA
describe the wobble base
the third base in a codon; leniency; can
1. normal bp
2. GU base pairing
3. inosinate can base pair with A, U, or C
describe tRNA
deliver amino acids to ribosome
cloverleaf secondar structure
anticodon runs antiparallel to codon
rigid L tertiary structure
invariant bases for structure; variant bases for amino acid pairing
aminoacyl-tRNA synthetases
different ones for each type of amino acid; how the amino acid binds to a certain tRNA; only certain tRNA with the right variant basescan bind to the right enzyme making it sequence specific which amino acid is binding
uses ATP and MG2+
what are ribosomes made up of
65% rRNA and 35% protein by weight; rRNA acts as an enzyme to catalyze reaction & Proteins there for structure/ shape
what is the function of ribosomes
Ribosome catalyzes formation of peptide bond
describe the binding sites in ribosomes for tRNA
A site: charged tRNA enters ribosome
P site: grow peptide chain
E site: exit/ base pairing weak here so uncharged tRNA leaves
polysome definition
multiple ribosomes bound to a single mRNA
describe translational initiation in a prokaryotic cell
initiation factors act on 30S ribosomal subunit (small subunit)
IF 1 blocks premature tRNA from binding to A site
IF 2 blocks premature binding of big (50S) subunit
mRNA binds 30S subunit through base pairing and the SHine- Dalgarno sequence
AUG start codon arranged in P site
shine dalgarno
ribosome binding sequence (RBS) where ribosomes bind to initate translation, base piars t o16S rRNA within 30S
what is the first amino acid in prokaryotic protein to bind
fMET, bound to IF-2-GTP and escorted in P-site, binds to start codon to begin translation
how does the translation initiation complex finishes in prokaryotes
IF-2 hydrolyzes the GTP resulting dissociation of all IFs; 50s subunit binds forming the complete 70S ribosome
A site is vacant; P site has fMET, E site is vacant
describe translation elongation in prokaryotes
Elongation factors (EF) act on 70s ribosome; EF-Tu-GTP binds charged tRNA and delivers it to ribosomal A site; anitcodon and codon pair while the actual charged tRNA is blocked by tetracyclines binding to 30S subunit
hydrolyzes EF-Tu-GTP and exits
A site has charged tRNA
P site with fMet
E site vacant
describe peptidyl transferase
23S rRNA
frree amino grop on actyl-tRNAin A site attacks fMet in P site, fMet is transferred to new chain in A site
how is peptide transferase activity regulated
chloramphenicol binds 50S (23S rRNA) to repress it
describe what streptomycin does
change shape of rRNA in 30S subunit, cauing a misread of mRNA
describe translation termination in prokaryotes
stops when stop codon is reached, the ribosome pauses to wait for a charged tRNA that will never come…
Release factor (RF) sees stop codon and binds to A site, activate peptidyl transferase, hydrolyzing peptidyl-tRNA bond to end translation
what are the major differences in eukaryotic translation initiation
eIF2 brings Met to 40S subunit BEFORE mRNA arrives
eIF4 binds to mRNA cap to brin git to 40S
scans for mRNA start codon within a Kozak sequence
60S binds to complete 80S ribosome
describe the role of ubiquitin in protein marking
polyubiquitin (a chain of ubiquitin) is used as a marker for protein degradation; covalently attached to target proteins
recognized by proteasome and digests the protein
describe a mutation
an accidental change in DNA sequece that may affect the sequecce of a protein encoded in an mRNA
nonsense mutation
introduces a stop codon
missense mutation
replaces one amino acid with another
silent mutation
changes the DNA sequence without altering the encoded protein
frameshift mutation
caused by insertion or deletion within a coding sequence but not in a multiple of 3 bases; changes reading frame
how can mutations arise
spontaneously or by environmental factors
what are the types of DNA damage
Deamination, oxidation, depurination, alkylation, thymine dimer, and DNA strand breakage
describe deamination of DNA Bases
causes C-> U and 5-meC-> T ( need 5-meC for the regulation of transcription); can be sped up with sodium nitrate/ nitrite
occurs spontaneously or from environment
describe oxidation of DNA
reactive oxygen species (ROS) generated by respiration, hydroxide free radical inserts into either G or T which changes the base pair, can result in strand breaks
depurination
caused by the hydrolysis of g;ycosidic bond linking purine base to sugar-phosphate backbone
yield abasic site (site without base)/ “AP” site (site without purine)
more likely to occur for purine than pyrimidine bc glycosidic bond is weaker
describe alkylation of DNA
alkylating agents covalently modify bases in DNA; spontaneous alkylation by SAM of G -> 7-methylguanine; distorts the DNA double helix; creates a kink to hard to work through
ex. sulfur mustard
describe thymine dimer
UV common cause to form cyclobutane ring between two adjacent pyrimidine rings; can create thymine dimers that kinks the axis of DNA helix
scribe DNA strand breakage
can be caused by ionizing radiation from sources such as cosmic rays, x-rays, and radioactive materials
describe DNA strand breakage
can be caused by ionizing radiation from sources such as cosmic rays, x-rays, and radioactive materials
can be single strand break/ nick or double strand and staggered
what is the general pathway of dna Repair
- recognize damage
- remove damage
- resynthesize DNA -DNA Pol 3
- ligation of loose ends -DNA ligase
must occur beofre replication so it doesn’t result in a permanent mutation
hormones
molecules produced and secreted by one cell type and transported to another to elicit a response; can be polypeptide, steroid or other, active at low concentrations and often subject to feedback regulation
growth factors
signaling proteins that act as mitogens to stimulate cell growth and proliferation of a target cell, also active at low concentrations
receptors
target cell proteins that bind growth factors and hormones with high affinity and specificity
signal transduction
the process that converts the binding of a signaling molecule to its receptor to elicit a response
second messengers
agents of signal transduction within the target cell
how does a cell go through the cell cycle/ know when to enter the next phase
utilizing intercellular communication via signaling molecules and receptors, growth factor and hormone induced signaling are used to produce cell cycle proteins that mark the transition into a new phase
how do steroid hormones communicate to a cell
steroid hormones are hydrophobic, they are able to easily pass through the cell membrane; they bind to nuclear receptors inside the cell; receptor becomes transcription factor that binds to its cis acting elements
how do peptide hormones communicate to a cell
because peptide hormones are proteins, they use cell membrane receptors to send their message which releases signals to get to trans acting factors in the cell; target GPCR and RTKs
what changes does a growing cell experience
altered gene expression
increased translation
increased nutrient uptake and metabolic rate
altered morphology
describe the steroid hormone example
estrogen = steroid
estrogen receptor = ER; estrogen response element (ERE) = gene sequence that ER binds to
ER exists in monomeric state, but when is bound by estrogen, enter a dimer state. receptor is translocated to the nucleus where it interacts with the coactivator proteins to recruit transcription machinery
describe the importane of JUN
JUN is a gene that is a subunit of the AP-1 TF; produced to regulate estrogen production;
describe the importance of FOS
FOS needs to be expressed for AP-1 to form ; expressed through peptide signaling
describe the importance of AP-1
activating protein 1, essential activator for many genes to progress through cell cycle
describe RTKs
receptor tyrosine kinases
receptor that is also a kinase that phosphorylates tyrosine
When protein signaling molecule binds, conformational change in receptor related to inside of cell; kinase domain phosphorylates tyrosine (itself)
RAS-dependent Pathway
a result of a peptide hormone response; RAS = on/ off switch for growth
when on, IGF-1R autophosphorylates; phosphorylated tyrosine = docking site;
SOS1 binds to phosphorylated tyrosine and activates guanine nucleotide exchange facotr (GEF) which will activate RAS
RAS-GDP bound when not dividing but GEF binds to RAS to make it into active form RAS-GTP
RAS-GTP + RAF activates protein kinase activity, RAF -> MEK -> ERK-> transcription factors
ELK-1 binds to SRE, turning on FOS
turn off by GTPase activating protein (GAP)
what is a cyclin
essential regulaotory subunit required for cyclin dependent kinase (cdk) activity; produced and degraded through cell cycle; active cdk phosphorylates
what phase matches with the presence of which cyclins
G1: cyclin D and E
S/ DNA replication: cyclin A
mitosis: B
what must be present for a cell to divide
constant signaling/ actation/ production of JUN, FOS, AP-1, cyclin D, and cyclin E; build up enough kinase activity to get past restriction point
what is the importance of cyclin D
denotes start of G phase, CDK4 or CDK6, active kinase phosphorylation in order to move cell into S phase
activates transcription factors to produce cyclin E
E + D triggers A to start S phase
upregulated by AP-1 and MYC to kick start cell cycle
describe the negative regulation of E2F/ Rb
cyclin D and E hyperphosphorylates Rb to release it from E2F; E2F is an activator responsible for replication in S phase; release of E2F creates an activator in late G1 to promote cell replication
Rb is transcriptional repressor
what initiates replication
in S phase, initiated by phosphorylation of complex proteins by cyclin A/ cdk 2 - phosphorylates origin of replication
what does replication initiation being enzyme catalyzed mean for the process?
many origins can be activated very rapidly
what is cancer
a disease resulting form an accumulation of mutations and epigenetic modification that favor cell growth; can be invasive (break connective tissue) or metastatic (travel in bloodstream to other parts of the body
what is a secondary tumor
same cells from an initial tumor that grow in a different area that repress the organs and eventually kill them
where is cancer more prone to develop
organs that require regular replenishment (skin, blood etc) but less likely in places that don’t replenish (neurons, heart cells)
where do the mutations that lead to cancer come from
errors in normal function, spontaneously, exposure to carcinogens/ radiation, genes, epigenetic changes/ change of gene expression
proto-oncogene
normal cellular gene that encodes a protein promoting cell growth and proliferation therefore has the potential to be an oncogene
oncogene
mutant form of porto-oncogene that favors excessive growth; just needs gain of function in one allele to activate accelerated growth
what is activation in terms of cancer biology
mutation that converts a proto-oncogene to oncogene; 3 ways Overactive RAS dependent pathway, amplification of Her2 Gene; chromosoal rearrangements
tumor suppressor gene
encodes a protein that inhibits cell growth; loss of function mutations favors growth
what causes a normal cell to turn into a tumor cell
cancer = disease of genome; needs more than 6 changes affecting proto-oncogene and tumor suppression gene
what is tumor profiling
cancer diagnostic tool where it samples the level of expression from cancer-related genes
use info to see what mutations are prevalent and which tissues to target in treatment
describe how an overactive RAS Dependent pathway can lead to activation
the genes encoding proteins along RAS pathway are porto-oncogenes; gain of function mutations will generate oncogenes
ex. gain of function mutation in Ras gene results in a RAS protein that interacts with RAF in the absence of growth factor (overactive)
describe how a missense mutation can lead to a RAS oncogene activation
RAS genes often mutate at codon G12 (for glycine) but goes from GGT-> TGT to produce a cytosine/ protein that cannot hydrolyze GTP trapping Ras protein in the signaling conformation (no GTPase activity)
describe how the amplification of the Her2 Gene can lead to cancer
HER2 is a receptor tyrosine kinase (RTK) that can activate the RAS-dependent pathway; mutation = amplification of Her2 Gene causing an over expression of a protein/ RAS-dependent pathway
what is amplification
an increase in gene copy number as the result of a localized error in replication along a chromosome
how does herceptin work
target Her2 positive cancers cells and binds to prevent Her2 from dimerizing/ activating and RAS cannot run; modern chemotherapy has improved survival rates
