Flashcards in Biochemistry Deck (163):
What is the genome composed of?
-all of the DNA in all cells
-includes coding and and regulatory sequences
-23,000 coding genes
-20% of the DNA contains genes
What is the transcriptome composed of?
-all of the transcripts (introns included)
-DNA regulatory elements not included
What is the exome?
-the RNA after processing!
-contains protein coding and regulatory sequences
-missing the introns!
What is the proteome?
RNA---> the proteins that reuses from this in a given cell
-500,000 different protein products
What is the metabolome?
-the metabolites of proteins that are produced from cellular processes.
-often can look at this and diagnoses diseases
What is the central dogma?
basically it is the flow of information of the -omes
-this is version 2.0
genome--> transcriptome--> exome--> proteome--> metabolome
What is the mnemonic for the nucleic acids?
PURe As Gold= PURines: A/G
CUT the PY= PYrimidines: C/U/T
What is the difference between a nucleoSIDE and a nucleoTIDE?
Nucleoside: have the sugar and the base
Nucleotide: has the sugar, the case, and a covalently bound phosphate group (up to 3)
What kind of linkage connect the DNA/RNA sugar to the base?
What is the primary structure of nucleic acids?
5'--->3' bases that are linked together by a phosphodiester linkage
RNA or DNA
Which is more stable? Why?
DNA is more stable due to the DEOXY at the 2nd carbon.
RNA has an OH at the 2nd carbon which can automatically cleave the phosphidiester bonds in aqueous (basic) solutions make it less stable. As a result of the decreased stability, RNA has a shorter life span.
**this is why DNA is the molecular library of life (longer life span!)**
What is the secondary structure of DNA?
-5--->3 phosphodiester linkages
-bases face the interior (where they hydrogen bond with one another)
a. 3 for G-C (higher MP and stability)
b. 2 fot A-T/U
What 2 things stabilize nucleic acid secondary structure?
1. hydrogen bonding between bases
-strands must be antiparallel for this to happen
2. base stacking
-the pi orbitals overlap and interact with one another. this keeps the water out from the interior of the structure and stabilizes the DNA
Different forms of DNA... what are they?
B form is the prototype form. It has MAJOR and MINOR grooves
-this form alternates between the A form (rigid) and Z form (gene silencing)
-different proteins can access different forms
-dynamically changes between these 3 types
What is characteristic of the A form?
-shorter more compact
-more bp between each turn
What is characteristic of the Z form?
-more stretched out
-has major and minor grooves
-GC rich---> methylation! so can get gene silencing
What is the tertiary structure of DNA?
supercoiling [relaxes molecule]
-if we overwind it then can denature due to weak regions (A-T rich) and make single strand---> activates replication system [happens in bacteria]
-mostly found in the B form
-essential for packing and stabilization with nucleosomes
What are denaturing agents of DNA?
3. chemical solvents
4. chemical modification (formaldehyde)
*primary structure stays intact!
*if denature by using 1-4 water rushes in and breaks h-bonds forcing strands apart
Why when you add formaldehyde to a dsDNA does it not reanneal?
It will form a COVALENT bond with the N at the 2 position and block G-C interactions
-therefore it is a carcinogen!
-used in carpets, plywood, and fiberboard (new home smell)
What types of interactions happen at the major groove of DNA?
transcription factors bind here
What types of interactions occur at the minor groove?
DNA-protein interactions with the histones.
-Lysine (K) + Arginine (R) interact with the negatively charged DNA backbone
-this is an electrostatic interaction
What is a nucleosome?
histone octomer + negatively charged DNA
-this is the primary fundamental unit of chromatin
-nucleosomes also have protein--protein interaction when they stack on top of one another and interact via the polypeptide domains that are sticking off of the particles
What is the H1?
H1 is the histone that binds to the linker DNA and the nucleosome... stabilizes the structure
Higher orders of chromatic structure are stabilized by...
aside from histones...
-interaction with scaffolding proteins and other domains has a large effect on the stability and structure of chromatin
What is epigenetics?
Changes in gene expression or cellular function caused by mechanisms other than changes in the DNA sequence
SAM--> attaches methyl groups to Lysine on histones
Histone Methylation Mostly Makes DNA Active
it blocks the electrostatic interaction and makes DNA more diffuse for gene expression
a. until chromatic structure is changed which leads to remodeling and reversed
HDAC---> removes the acetyl group
histone Acetylation makes DNA Active (increases gene expression) by relaxing DNA coiling and allowing for transcription
1. tags old denatured proteins
2. modifies proteins to give complexes an alt. function
-loosens up the chromatin --> increasing gene expression
-poly ubiquitinylation leads to destruction
-some patterns are inherited
-directs mis match repair in damaged DNA (distinguishes between old and new)
-plays role in barr bodies (inactive X)
-there is a normal level of methylation but...
-environment can cause methylation!!!!
SAM--> adds methyl groups
CpG Methylation Makes DNA Mute
if CG rich in promoter region and methylated this leads to a change in the DNA structure (to Z form) or by recruiting HDAC activity and shutting down gene expression
What are barr bodies?
inactive x chromosome
-due to methylation condensation of the DNA
5 aza cytosine and its use as a chemotx
-blocks methylation of cytosine --> hypomethylation
-covalently binds enzyme to DNA so no methylation can occur
-this is a nonspecific process though and can either activate genes that were suppressed such as oncogenes or it can suppress genes that are important like tumor suppressor genes
ex. in class of Elliots father in law with bladder infection over Rx this drug (which is actually a last line/try drug) and he developed AML and died 2 years
What is fragile X syndrome?
cause: trinucleotide repeat (CGG) on FMR1 gene --> shut down in gene expression
-x linked: early on no symptoms till get to 200 repeat range
-most common form of inherited mental retardation
the repeat leads to methylation which then condenses the chromatin leading to the morphology of fragile X.
mRNA gets suppressed and proteins for normal maturation and development are not made
CGG--Chin (protruding) Giant Gonads
DNA Replication is ___ and ___
Semi conservative : because it uses a parent strand and then makes a new strand off of this template
Semi-discontinuous: because the leading strand is continuous and the lagging strand is discontinuous made up of Okazaki fragments
How does DNA polymerase add nucleotides to the DNA chain?
It is an enzymatic rxn
Things required for reaction to proceed:
1. ssDNA template
2. RNA primer
3. Free 3-OH end
4. 4 dNTPs
6. Appropriate environment
5 end DNTP is always added to the 3 OH END. So synthesis happens in the 5--> 3 direction
**hyrdolysis of PP drives reaction forward and is irreversible
DNA pol 1
DNA pol 1 - repair enzyme, has polymerase and exonuclease activity in both directions
-fills in the gaps 5--3 polymerase activity
-removes Okazaki/RNA primers
after DNA pol 3 this kicks in to remove primers 5-->3 exonuclease
DNA pol 2
-has polymerase activity
-no 5-->3 exonuclease BUT has 3 to 5 which allows it to look at what it's putting down
-acts as a back up for DNA pol 1 (ssDNA gap filling repair enzyme)
DNA pol 3
-aka replicase-- does DNA synthesis in bugs
-also has exonuclease and repair activities
-has a 4 beta subunit domain that acts as a sliding clamp for the repair mechanisms
DNA polymerase in eukaryotes
-no exonuclease activity (this is taken over by other associating proteins)
-has a PCNA trimer (proliferating cell nuclear antigen) its the sliding clamp that is required for DNA replication and damage repair.
-much slower rate of Replication because of the proteins that assure for accuracy
Fidelity of DNA replication
-in both prokaryotes and eukaryotes DNA rep has high fidelity.
-it is higher in eukaryotes
-high fidelity in the ones that are constitutively expressed
and LOW fidelity in those that are activated by damage (aka UV light)
What is the origin of replication? prok. vs euk.
-specific DNA sequence (usually AT rich)
-has recognizable proteins attached to it
in prok. the supercoil of AT rich regions causes denaturing which forms an alternate structure that proteins recognize and then bind to---> replication
in euk. the AT rich regions lead to the A form being formed. Proteins recognize this form and then start recruiting other enzymes necessary for replication to begin.
what is a replicon?
=entire region of DNA that is replicating
-in prok. they have 1
-in euk. there are many
what is a primosome?
= RNA polymerase (primase) + assoc. DNA sequence that it is attached to
the DNA--protein complex!
what is a replisome?
=DNA polymerase and all of the assoc. proteins (each have specific function) necessary for DNA replication
-this complex replaces the primosome
What does DNA helicase do?
it uses ATP to cause a conformational change that then leads to the unwinding of the dsDNA.
What is the phenomenon of looping out during DNA synthesis?
-this reduces structural strain of the opposing polymerase action on each of the strands
-the LAGGING STRAND wraps around the replisome making replication occur in the same direction
-length of loop=length of okazaki fragment
-each loop needs new RNA primer
aka trombone model
What does DNA ligase do? What is different in prokaryotes vs euk?
ligase uses: prok-use NAD auk-use ATP to energize the enzyme so it can add to the 5 end and seal the okazaki fragments together
What are some examples of gene amplification due to triple repeats?
1. huntingtons- CAG (glutamine) repeats
2. fragile X- CGG
stable CG rich stem loops form over time and replication and cause the disease state in offspring
topoisomerase and the different versions
topoisomerase detects supercoiling strain and cuts dsDNA to reduce the strain. Topo also re-ligates the strand after it cuts it
topo I- cuts one strand
topo II-cuts 2 strands **requires ATP for activity**
What is ciprofloxacin used for?
inhibits topo 2 by blocking the phosphodiester link
what is etoposide?
inhibits topo 2.
-can be non specific and damage other fast growing cells
What causes the termination of replication?
the replisomes slowly approach one another and eventually crash into each other. this causes the replicon to fuse --> patch repair by DNA pol
What are telomeres? And how do they work?
-necessary for chromosomal stability
-increasing age... shorter telomeres
-produced by telomerase (a reverse transcriptase enzyme)
TER= RNA sequence as template AAUCC=humans AACCC=tetrahymena
TERT=(telomerase) reverse transcriptase RNA-->DNA
-redundant sequences --> fold onto each other --> form tetra helix =seals chromosome end!!! stability!
What are the 4 types of DNA damage?
1. physical damage
2. conformational changes
3. DNA rearrangement
4. chemical modifications
-donate methyl of ethyl
form unstable carbocations --> electrophillic attack --> covalently binds to DNA
-this can result in change in the genetic stability, chromatin structure, alteration of epigenetic regulation
a second type of DNA damage which leads to DNA point mutations.
-adenine to hypoxanthine to cytosine can be repaired
-cytosine to uracil can be repaired
BUT deamination of 5methyl cytosine to thymine is not recognized as damaged and gets incorporated into DNA --> GI cancers HOT SPOT BC IN TRANSCRITIONAL REG. REGION
*sodium nitrates in (ham) and sodium bisulfites (wine) preservatives can INDUCE oxidative deamination*
Intercalation and the drugs used to tx it
-change in DNA structure
-a planar aromatic structure slips into DNA structure and goes undetected... this causes problems when DNA replication tries to occur leading to whole sequences being left out or mis-pairing reactions
Rx: actinomycin D and psoralen
What are benzopyrene and aflatoxin? and what do they do to DNA
-benzopyrene often found in grilled foods or from things that combust without sufficient oxygen (forest fires)
-aflatoxin is from mold on grains
p450s activate and elminate these. BUT! if superinduced and system is overwhelmed they intercalate and covalently bind to DNA -->large disruption of chromatin structure --> activated SOS repair
physical damage to DNA
-induced by UV rays --> causes kink --> disruption of helix
-if these dimers accumulate and are left unprepared then this region will be completely skipped or the replication system will insert random nucleotides to try and fit in the sequence... large mutations.
What is xeroderma pigmentosum?
-mutations to the DNA repair system for thymine dimers on chromosome 9
-this leads to a host of skin issues including cancers, retinal degeneration, neurological abnormalities
What is the mechanism of Direct Reversal of DNA Damage?
-either replace directly a missing base
-photoreactivation of thymine dimers using the same UV light that caused the mutation
-oxidative deamination (a good one) by an enzyme that recognizes the bad base
What is excision repair? long patch vs. short patch?
1. system recognizes the damage
2. 5' endonuclease is initiated
3. exonuclease activity through the damaged region
4. DNA polymerase lays down new sequence
5. ligase seals the gap of the backbone
short patch=short damage 10-30 nc
long patch= extensive damage 200-300nc
what is mismatch repair?
-this is a type of excision repair that looks after newly synthesized strands
-parental strand is methylated... the template strand is not methylated therefore the mismatched base will be recognized on the template and be replaced
-there is a delay in methylation due to this so things can be edited
-once methylation of the template strand occurs mismatch repair stops
what is double stranded break repair?
-rad50 genes are important to stabilize ds break fragments (BRAC1/2 stabilize this)
homologous end joining uses the enzyme to repair with non damage DNA strand
non homologous end joining when proteins mediate the recognition and repair of the break and rejoining **this is subject to errors and mutagenic**
-occurs at specific sequences
-transposons and control gene expression from moving
-this is a highly regulated process
however... this can lead to change in stability, cell growth and maturation, and changes in normal physio --> cancer
what are cohesins?
-they hold the sister chromosomes near one another for recombination and dna damage repair (if needed)
-are important for repair and cell division
-acetylation of them is like histones where locks it in with the DNA and promotes synthesis
what are the error prone repair mechanisms?
2. excision repair
3. recombination repair
usually SOS repair
what is the Philadelphia chromosomes link to cancer?
-this is a translocation of a part of chromosome 22 to 9.
-this translocation results in a hybrid which drives proliferation of cells and no auto regulation of the protein
-this causes chronic activation which leads to CML
what does the gleevac drug due?
its a drug that binds to the translocation site only and competitively inhibits the activation of the activation site and no ATP binding.
-this leads to no activation of the signal transduction pathway and ultimately no leukemia
c-myc and lymphoma
c-myc is a protooncogene and is important TF for gene expression
-sometimes c-myc can undergo recombination with chromosome 14 (8-->14). this places it next to a heavy chain sequence that initiates the immune system...
-if the immune system is turned on then c-myc will actually be OVER enhanced and cause amplification of cell division which leads to overgrowth benign tumors "Burkitts Lymphoma"
faulty DNA damage repair can lead to this
describe the initiation of transcription
1. transcription factor binds to the specific seq. on major groove of the DNA (A form) and stays inactive until ENVIRONMENTAL change causes recruitment of proteins via a signal transduction pathway
2. TF gets turned on or off from this change. IF ON...
3. proteins get recruited (COACTIVATORS) including HAT to acetylate the histone and loosen up the DNA on the histone.
4. this loosening pushes nucleosomes out of the way uncovering a PROMOTER site
5. this now recruits more proteins to the site like RNA pol 2 to start site to initiate transcription
6. transcription begins on the strand while HAT and remodeling engines move nucleosomes out of the way
at the end of the system HDAC removes acetyl groups and the dsDNA rebinds
what are cis elements?
the DNA sequences:
what are trans elements?
transcription factors (proteins)
what are some key differences in prokaryote transcription?
-all done simultaneously with DNA replication in ONE compartment
-trans elements are called sigma factors
-no post translational modification
-degraded within minutes
-only ONE RNA polymerase (with several subunits)
what is a operon?
=a group of functionally related genes driven by a single promoter element
explain the lac operon
classic example of environmental influence on genetic responses
lactose is taken up into cell --> converted into allolactose -->binds to the repressor protein so it cannot bind to the operator to block transcription --> lactase is expressed
-the sigma factor can bind to the promoter element and initiate transcription
what is polycistronic mRNA?
=more than one gene product per mRNA
-the lac operon is polycistronic produces three protein products z,y,a
eukaryotic promoter elements
-they are arranged in very complex and precise ways in order to have control over gene expression under different environments
-have silencers and enhancers (ex. estrogen)
-these specific dan sequences of cis elements are NOT in the transcriptome or exome
-TATA box or CAAT box are good examples but there are others in addition that are essential to translation initiation
this is an example of transcription factor interaction driven gene expression.
a leucine zipper is a protein-protein interaction which causes dimerization of the protein which stabilizes the structure for other transcriptional factors and co-activators and the RNA pol complex
what could happen if there were mutations in the leucine zipper?
-blocking of dimerization---> altered gene expression and neoplastic transformations
why is the flexibility of DNA important for transcription?
it allows chromatin to move between forms for expression
-activator or inhibitor elements can be brought near the promoter (which is next to the ORF) and stabilize the enhancer or silencer which thus regulates gene expression
what are the 2 signal transduction pathways?
1. TF is bound to DNA in an inactive state and is activated by a signal that comes into the nucleus
2. TF is out in the cytosol and receives a message to become active
3. TF is in the cytosol bound to a protein waiting for message to break free from protein to become active and bind to the DNA sequence elements.
What does PKA and CREB do to gene expression?
CRE= cyclic AMP response element (DNA seq.)
CREB= CRE binding protein [latent and inactive bound to CRE]
CBP= CREB binding protein
phosphorylation of CREB --> CBP binding ---> recruitment of tfs on adjacent promoter region
--> RNA pol II--> gene expression!
**dont forget HAT and remodeling engine complex are necessary part of proteins needed for transcription**
1. most of them are on the DNA sequence in an inactive state waiting to be activated by estrogen to form a dimer and initiate the cascade if events that is transcription
2. some other estrogen receptors may be in the cytosol and are trafficked to the nucleus
-estrogen is synthesized in cytosol
enhancer of gene expression!
helix 12 and estrogen
when estrogen binds...there is a ligand domain known as helix 12 that undergoes a conformational change so that the coactivator and other proteins are able to bind to the correct site and initiate gene expression
what does tamoxifen do? What are problems with tamoxifen and rolxifene?
tamoxifen essentially works on breast cancer by blocking the conformational change of the helix 12 and not letting the structure dimerize and fold up... thus no gene expression.
-it is sensitive to alpha cells which are in breast but activated beta which is in ovaries which may cause cancer there
-the opposite is true for raloxifene... be careful when rx
RNA pol 1
*think of in order that they are used in RNA synthesis*
RNA pol 2
transcript: mRNA, snRNA, miRNA
RNA pol 3
transcript: tRNA and smallest rRNA
what is the elongation phase of transcription initiation?
once the RNA pol 2 lays down a few "tester" sequences and it is determined stable elongation phase begins and the stable RNA pol complex releases the TFs and proceeds forward.
RNA pol can act as helicase and topoisomerase for the coil
what are 3 RNA polymerase inihibitors?
antinomycin D: intercalation (prok/euk)--research and chemo
amanitin: inhibits pol 1/3--in death mushrooms
rifampicin: block RNA pol initiation and elongation--antibiotic
how does transcription terminate in prokaryotes?
1. RNA sequence structure dependent: the newly synthesized RNA forms a hairpin loop of lots go C-G rich sequence. Beyond this there is a UUUU sequence. This causes a stress and shear force that breaks the two strands and pulls of RNA pol
2. rho protein: basically attaches to RNA strand and uses ATP to pull the strand towards it until it reaches the RNA pol complex where it proceeds to pull the strand off of the polymerase
how does transcription terminate in eukaryotes?
structure dependent termination... hairpin loop!
-unique Pi linkage so that exonuclease can't degrade it
-regulatory for translational initiation
**this and poly A tail are essential for mRNA stability and regulation of translation**
a. protects from 3' exonuclease
b. sequence for binding proteins
-polyApolymerase synthesizes this RNA
mRNA is not straight it loops around at the end so as to protect the mRNA
-this is the SECONDARY structure of RNA
-introns contain splicing information to accurately assemble the final transcript
-splice sites at exon--intron interface are highly conserved
-splicing occurs in a step by step process
-it is done alongside snRNPs as they exit RNA pol
mechanism of splicing
1. transcript combines with snRNPs and other proteins to form the spliceosome
2. lariat shaped (loop) intermediate is generated
3. lariat is released to remove intron and join the 2 exon
-1st cleave is at the GU
-there is an conserved A in the middle of the intron slightly upstream from the 2nd cleavage site AG
-the intron is degraded
alternative splicing... what can it lead to?
-can be planned based upon environment and protein interaction OR can be caused by a mutation which forms a new splice site
-beta thalassemia is an example (altered oxygen binding)
-this is why have same orig. sequence but then different functions in different tissues throughout the body.
can lead to:
a. altered protein activity
b. disregulation of cell targeting and trafficking
c. different enzyme substrates and reaction rates
-acid hydrolysis of phosphodiester backbone
-degrades rogue RNA molecules (introns)
-intracellular in lysosome
miRNA and things it does
-does posttranscriptional modification of gene expression
-it is processed by DROSHA and DICER --> dcmiRNA-->ssMRNA --> binds to RISC
depending on the environment and proteins around...
a. RISC complex can block translation and save for later
b. RISC will bind with ARGO and degrade the mRNA in the mRNP complex
mRNA is scanned in the...
proteins are synthesized from the...
N terminal to the C terminal
what energy sources drive
a. aa binding to tRNA
b. protein synthesis
a. ATP charges the tRNA molecule to bind the AA
what is the start codon? what are the stop codons? why are they stop codons?
stop codons: UAG, UGA, UAA
*there are the stop codons because there are no associated tRNAs for these codons*
why is the third position known as the wobble position?
it is known at the wobble position because the tRNA doesn't necessarily depend on it for its binding. The first 2 nucleotides are more important for determining the binding.
-the 3 end is also less mutationally active. meaning that a change in this nucleotide will not have as big of an impact
the genetic code is ___ and ___.
degenerate: meaning the there may be more than one triplet code for an AA
redundant: there can be multiple codons for a given AA
why is the genetic code basically universal?
It is universal because the same codons "mostly" code for the same AA's. However, in some cases different organisms have a codon that codes for a different AA
for ex. CUG is a "weak" start codon in c-myc
What is codon bias?
-it is when an organism has a different preference of set of codons that code for a specific AA
-for ex. out of 6 Leu codons, maybe the organism only uses 4 making the polyp chain a different protein
-bias can lead to large problems in cloning genes from prok--> euk.
-codon bias optimizes the protein synthesis machine for that species
different types of mutations in protein synthesis:
insertion: frameshift or premature stop
deletion: frameshift/alt. sequence
nonsense mutations-premature stop
How does tRNA bind to AAs?
-twisted L confirmation is common to all tRNA's
-AMINOACYL TRNA SYNTHETASE binds a corresponding AA to the 3' end of a tRNA molecule
-this also has a proofreading mechanism that immediately hydrolyzes the incorrectly attached AA from the tRNA
-tRNA contains the anticodon to the AA codon sequence
**ATP DEPENDENT REACTION**
-major mass of any organism
prOkaryotes are ODD
Eukaryotes are EVEN
-these are bigger which means they have more interactions and more regulation on translation
what are the secondary and tertiary structure of ribosomes?
-it is more of a globular structure
-there are many intra-molecular interactions (stem loops) and stabilizing binding pockets and domains that stabilize the overall structure
describe initiation of translation
there is a consensus sequence that binds to the antiparallel sequence of this on the small subunit of the small ribosome. Once the ribosome binds to this sequence it scans the mRNA from 5-->3 until it comes upon the AUG start codon.
1. IFs are bound to the small ribosome
2. initiator tRNA (that is bound to GTP and start AA) scans the consensus sequence until it reaches the start codon
3. it binds and GTP is hydrolyzed
4. IFs lose affinity
5. large subunit sits on small subunit
6. initiator tRNA binds to the P site
-initiated my a formulated methionine
-shine delgarno sequence is the identifying sequence for initiation
-kozack consensus sequence like shine delgarno but this INCLUDES the AUG start codon
-methionine is the start
-lots of secondary structure in 5UTS region of mRNA--regulation for translation
-eIF4 complex=helicase to disrupt these secondary structures
what is pateamine A?
RNA helicase blocker to block protein synthesis in cancer cells
describe the process of elongation
an AA-tRNA complex binds to the EF-Tu elongation factor which brings the correct tRNA to the A site. GTP is then hydrolyzed. A peptidyltransferase then forms a peptide bond between the AA in the A site and the growing chain in the P site. This moves the chain to the A site momentarily. Then EF-G (structural mimicry to a tRNA) with a bound GTP becomes hydrolyzed and causes a shift in the ribosome by 3 nucleotides moving the chain back to the P site. [translocation]
-empty tRNA's are then moved to the exit site with EF-G induced translocation... this tRNA is then recycled and reused
what is a ribozyme?
it is an RNA molcule capable of acting as an enzyme
-peptidyltranferase and formation of the peptide bond is this
describe the process of termination
a stop codon appears in the A site and there is no associated tRNA for it. A releasing factor (RF) binds to GTP and sits on the A site. GTP is then hydrolyzed and the synthesizing complex dissociates.
-polyp chain released
*the RF undergoes a confirmation change after hydrolysis of GTP which drives the ribosomes apart
elongation in eukaryotes
-polysomal: can have more than one complex of ribosome on mRNA chain
-the ribosome do dissociate upon termination but stay close by to be reinitiated and start translation on the same strand=efficiency!
there can be a number of secondary structures in the mRNA that are intentionally there to slow down translation of a protein (important for c-myc)
-since euk. mRNA is more of a circular structure the poly A tail is very important for stabilizing the mRNA as a mRNP complex
examples of c-myc and translation
-start is in exon 2
-exon 1 usually not translated and has a secondary structure that super downregulates expression of c-myc which is good because don't want a lot of it because it can lead to neo-plastic formations
-translocations/rearrangements/recombo of c-myc can lead to regulatory loss and ^^expression
-c-myc also has an alt. start sequence
What are internal ribosome entry sites?
IRES are secondary structures in the mRNA that act as "landing pads" for ribosomes to bind to
-no mRNA scanning is required therefore inhibition of translation can be circumvented by these things
-viruses can take advantage of this
what does polio have to do with IRES?
the polio virus goes through a process of making its own proteins
-viral proteases cleaves the binding proteins for initiation of translation (no scan... no host protein)
-it also forms IRES which ribosomes can bind to and translate the viral mRNA
-human cells die due to the inhibition of protein synthesis
-this detoxifies cells and the body of iron and potential free radicals
-example of environmental effects on translation
-example of IRES: hairpin loop in 5UTS of the mRNA for ferritin binds the proteins
if Fe is LOW-proteins bind to mRNA and BLOCK translation
if Fe is HIGH-proteins are released from the mRNA and permit translation
transferrin mRNA regulation
secondary structures are in the 3UTS region.
-aconitase (from the krebs cycle) which has Fe-S that stabilizes the secondary structures and prevents its degredation
if HIGH FE in the cell... the aconitase is fully modified with the Fe-S center... therefore it cannot bind to the structure and stabilize it therefore allowing for degradation of the mRNA
if LOW FE in the cells.. the aconitase loses the Fe-S center and can not bind to the secondary structures... allowing for mRNA translation and transport of Fe around the body using transferrin
what is an example of post-transcriptional RNA editing?
-there is a specific enzyme in the liver which can cause deamination of a cytosine to a uracil
-this can lead to production of a more truncated protein leading to a different function
apoB48 is in the intestines-chylomicrons (dietary fat delivered to body)
apoB100 is in the liver and is the original mRNA that is edited to make a different protein. LDL-lipid trafficking is its function
what does erythromycin do?
binds to 50s subunits and inhibits translocation in prok.
what does ADP ribosylation do?
-an ADP group gets transferred from NAD and covalently binds it to the EF-2... blocking translocation and ultimately resulting in cell death
why is ricin so dangerous?
RIP: ribosomal inactivation protein
-brought into the cell by endocytosis and cause depurination
-this leads to blocked protein synthesis and cell death
-general protein degradation system
-optimal ph ~5
-activity is localized to the lysosome
-target sequences target the system to the lysosome
what is the difference between mono and poly ubiquitinylation?
-mono means gene expression and allows for a structural modification of histones to lose up DNA
-POLY is for S26 directed proteolysis
i. specific sequences are recognized and are tagged for degradation
What are the various ubiquitinylation complexes and what are their mechanisms? What AA is targeted on the protein?
E1: see sequence and charges the enzyme by adding a ubiquitin
E2: ubiquitin is transferred again to another cysteine residue
E3: the ubiquitin is transferred to a LYSINE residue on the target protein
Once proteins are tagged with ubiquitin... how are they targeted for degradation?
1. escorted by chaperone proteins to the S26 proteasome
2. target protein is degraded into smaller polyp chains to be broken down further into AA for re-use
*the ubiquitin is reused as well
**ATP DEPENDENT PROCESS**
What does bortezomib do?
S26 protease inhibitor
-blocks rapid turnover of apoptosis inducing proteins in cancer cells
difference between cytosolic ribosomes and membrane bound proteins that they make?
A. cytosolic ribosomes make cytosolic proteins that are targeted to organelles by chaperones
B. RER ribosomes synthesize proteins that are directly inserted into the ER membrane or are extruded through a vesicle--targeted for secretion from the cell
what is post translational targeting?
-this occurs once protein synthesis AND chemical modification have occurred
-involves chaperone assisted transport to cytoplasm, nucleus, mitochondria, and peroxisomes
-usually the protein has a specific target sequence that binds to the protein and instructs it where it needs to go
what is co-translational targeting?
-this is the secretory pathway
-targeted to golgi, lysosomes, plasma membrane, and or secretion
-this is the more common one!
what are the sequence of events that occur in membrane and secretory protein synthesis?
**see drawing for further detail in notebook**
1. AA exiting a ribosome form a signal sequence
2. sequence recognition particle (SRP protein) is near exit site of ribosome and BINDS to the signal sequence. (this halts synthesis)
3. SRP recognizes docking protein on RER **GTP is hydrolyzed to open the channel in the RER membrane to extrude the chain thru**
4. SRP is released and recycled
5. protein synthesis is resumed. Signal sequence is cleaved off
6. synthesis goes until stop codon (post trans modification occurs form here or fusion with other cells)
what happens to proteins that are integral membrane proteins?
these have an additional internal AA sequence (STOP TRANSFER SEQUENCE) that stops peptide chain elongation
-reconfirmation occurs so that the growing portion of the protein can be in the membrane
what is a translocon?
protein complex that is associated with translocation of polypeptides across the membrane
what are n-linked glycoproteins?
*n glycosidic bond to asparagine
-cell surface receptors
-extracellular enzyme activities
what are o-linked glycoproteins?
*o glycosidic bond to serine or threonine
-joint and organ coverings
-coordinate lots of hydrogen bound water--shell of hydration!
where does glycosylation occur?
protein glycosylation and dolichol phosphate
-n linkages typically use dilochol phosphate as its core hydrophobic molecule
-this takes place in the cytosol and is flipped into ER where it is attached to the corresponding side chains
-o linkages have a different core oligosaccharide that is built one sugar at a time
what are lipid rafts
-areas in the plasma membrane the are highly rich in lipids (cholesterol) and proteins
-they are areas that are assoc. with clustering of membrane proteins and membrane endocytosis where lots of vesicles excrete their stuff
-enhanced bioactivity of growth factor receptors, and other stuff sit here
moves vesicles from the golgi retrograde back to the ER
forms secretory vesicles moving from RER to Golgi
involved in pinocytosis at the plasma membrane
forms both secretory(exo) and endocytotic vesicles at the plasma membrane and lysosome
-on membrane areas that are rich in protein
-connected to adaptor proteins
-activated by phosphorylation
what is the process of vesicle fusion with the target membrane?
1. once are at target membrane coated proteins go away
2. this exposes V snare on the vesicle
3. V snare interacts with T snare which is on the vesicle
4. the 2 membrane fuse together by a protein called SNAP-attacted to T membrane
5. Rab GTPase hydrolysis causes delivery of contents or fusion with the target membrane
V/T snares are specific for different organelle targeting
ex. of procollagen synthesis and secretion
-vitamin C dependent process
-the protein is continuously modified in the RER and golgi until it is expelled by a vesicle to head for the plasma membrane
-in this whole process the collagen fiber has made a triple helix for itself
-once at the membrane it is expelled in the the EC fluid where it is further processed to generate tropocollagen
adaptor protein on clathrin
-targets vesicles to the plasma membrane
-once inside the vesicle clathrin is removed and an endosome is formed and targeted for lysosome for degradation
targets clathrin vesicles to the lysosome
how are vesicles trafficked by microtubules?
remember microtubules are made of a/b tubular and they have polarity (+/- ends)
-dyenin and kinesis bind to vesicles and move in separate ways
-ATP is hydrolyzed inducing conformational changes in order to move down the microtubule
how are vesicles trafficked by microfilaments?
-movement is similar to that of microtubules where ATP hydrolysis moves the structure along with vesicles
what is the process of protein targeting to the mitochondria?
most mitochondrial proteins are actually made from nuclear genes
-the protein has a tagging sequence [mitochondrial localization sequences] on the N terminal end that is identified by a chaperone
-the chaperone takes the protein to the mitochondria where ATPase denatures the structure
-this denatured protein is then strung thru TOM/TIM pore into the mitochondria
-at the aid of heat shock proteins it is refolded once inside the mitochondria (ATP needed)
what happens when a protein needs to be directed to different parts of the mitochondria? what are the proteins needed for each?
there are SPECIFIC membrane targeting sequences on the protein that direct it
tiny TIMS= intra mito membrane
TIM22 + carrier protein= inner mito membrane
TOM + SAM + tiny TIMs=outer mito membrane
nuclear protein transport--describe the process
-bind to nuclear localization signals NLS
-the importin a/b dimer complexes with the protein and passes it through the nuclear pore
-RanGTP binds to B and dissociates it from the complex
-NLS-and a lose affinity and dissociate as well
-Ran GTP + CAS + a get exported together out of the nucleus for recylcing
-RanGTP +B export as well