DNA, Gene expression and protein synthesis Flashcards
DNA structure
- 3-D structure
- 2 helices wrapping around each other
- the surface has major (wider spiral) & minor grooves (narrow spiral)
- the size of the groove affects access to bases within the helix
DNA & drug pharmacology
- a target for chemotherapy drugs through various types of interactions
- strand breaker: bleomycin (both helices break)
- non-covalent interactions: other interactions may occur too (not chemically interacting but fitting)
- covalent complexes: cis platin - platinum-based
non-covalent interactions with DNA
- intercalation between bases - doxorubicin (planar molecules fit)
fitting into (in space/opening)
- minor grooves - distamycin A
- major grooves - neocarzinostatin
- or spanning both - nogalamycin
*some do both
cis platin
*anticancer chemotherapy drug
- covalent binding –> specific to one strand (nucleotide-nucleotide or to protein)
- involving at least one guanine
- multiple ways of binding increase effectiveness as drug
types of covalent cis platin binding
*always to one guanine nucleotide
a) interstrand - consecutive nucleotides opposite strands, ~3-5% (one strand across another)
b) intrastrand - consecutive nucleotides, ~80-90% (2 covalent interactions on one strand - same backbone)
c) intrastrand - non-consecutive, nearby nucleotides, ~3-5% (skip residue)
d) either strand - guanine & protein, ~3%
How & why is cis-platin binding detrimental to cancer cell? Are only cancer cells affected?
- covalent bonds across helix or along one strand block RNA or DNA polymerase decreasing DNA replication or mRNA production for cell growth
- DNA is a template for polymerase
- slower growing normal cells likely less affected than faster replicating/faster-metabolizing cancer cells
- chemotherapy has a negative impact on cancer cells
*nonselective so not only cancer cells are impacted (all cells that replicate/transcribe DNA)
DNA replication overview
- replication events/requirements
- chromosome ends (5’ –> 3’ rule) & unwinding (topoisomerase)
- possible targets for chemotherapy
- topoisomerase unwinds double strand DNA
- 5’ –> 3’ rule because added onto exposed 3’ hydroxyl
- double helix slows down DNA polymerase so needs to be unwound by topoisomerase
the 5’ –> 3’ rule
- templates are anti-parallel
- RNA primer required to provide initial 3’ OH for DNA nucleotides add on
- replication proceeds 5’ –> 3’
- replication fork moves
- RNA primer (short stretch of RNA) binds antiparallel and daughter strand synthesis starts after on 3’ OH available for DNA polymerase
2 kinds of daughter strands
leading strand
- continuous synthesis (faster)
- one RNA primer and synthesis
towards the replication fork
lagging strand
- short, discontinuous synthesis
yields separate lengths of DNA
(Okazaki fragments) –> slower
- RNA primers removed by RNAseH
the enzyme, DNA nucleotides fill in
gaps, fragments joined by ligase - several priming events (slower)
- use 3’ end of RNA for DNA
ligase
*ligase completed
- RNA primers come off template before they are incorporated
- covalently ligated together for one covalent strand
- before they are not covalently linked
- when RNA primer removed, 3’ OH available to fill
a consequence of the 5’ –> 3’ rule
*leading and lagging strand
- chromosome ends (telomeres) shorten with repeated rounds of DNA replication
- single-stranded DNA template at chromosome terminus left after removal of RNA primer is degraded by exonucleases
- gaps remain unfilled and single-strand is degraded
- single stranded has no replication event
What are the consequences of repeated rounds of DNA replication?
*shortened each time cell replicates DNA
- both ends of the chromosome (telomere) are shortened
chromosome ends
- telomeres = ends of linear eukaryotic chromosomes
- protect & stabilize the internal section of chromosome
- conserved, short, highly repeated sequence
- ~6-9 ntds (similar but can vary)
- keep an eye on that TTA series
*do not contain any protein-coding but if degraded too much –> start to degrade protein coding region
telomere shortened too much
- cell stops replicating (loses the ability to replicate DNA)
- a sign of aging in cells
- single-stranded region degraded (telomere shortened)
- telomere shortening may = ‘biological clock’ of cellular age
- no mitosis but can still transcribe, etc.
end replication problem’ of DNA synthesis
- DNA polymerases need RNA primer to start replication
- 20-200 single-strand end left after removal of terminal primer
- single strand region removed by exonuclease & chromosome shortens
how to rebuild telomeres
*cell can slow progressive degradation if right enzymatic activity
- telomerase = catalyzes telomere lengthening (enzyme for mitosis forever)
- functions as reverse transcriptase synthesizing DNA from RNA template - contrast to typical DNA replication
- enzyme function requires protein and RNA subunit
- adds ntds to single-stranded overhang which becomes long enough to pair with usual RNA primer
- single-stranded portion now long enough to be primed by usual means
- long enough for daughter strand (but some shortening)
*balance between telomerase enzyme + DNA replication
telomerase structure
- RNA template (with uracil) in backbone and associated protein
- adds DNA onto 3’ hydroxyl
- temporarily/transiently base pairs
- template within (RNA) should extend strand - complementary to repeats making telomere
telomerase in normal cells
*good/bad news
- the majority of normal cells do not produce telomerase (somatic cells)
- consequences for tissue/organ
- telomeres shorten –> cell stops dividing –> replicative senescence
- accumulated tissue “wear & tear” without new cell replacements
- if re-extension of telomeres –> then continued cell replication
- cellular aging/dying
telomerase activity
- reproductive cells: moderate levels
- blood, skin, & gastrointestinal cells: very low levels
- tissues where replacement and renewal are critical
- some shortening still occurs because the level is too low
- ex. epithelial (no rep) in the villus of the small intestine for absorption
- crypt is the location for high replication (more telomerase) and lower levels of telomerase
- at the tip, cells are shed + replaced by replicative cells at the crypt (travel up)
- ex. skin cells have a low level of replacement = low telomerase
cancer cells and telomerase
- ~90% of cancer types have high telomerase levels
- telomerase levels increase from early to late-stage cancer
- telomeres maintained; cells divide & escape replicative senescence (no internal clock)
- the other ~10% of cancer cells maintain telomere ends
- alternative lengthening of telomeres (ALT) = sister chromosome serves as template in a process similar to homologous recombination
- extended telomere of matched chromosome
(t/f) telomerase & alternative processes may be anticancer targets to block chromosome maintenance
true
DNA replication as a drug target
- telomerase function blocked by “antisense” DNA
- telomerase function blocked by AZT
telomerase function blocked by “antisense” DNA
- restore the clock to stop replication
- complementary to the RNA component of the enzyme (plug up –> preoccupied with something else)
- inactive telomerase complex (DNA+RNA+protein)
- RNA not available as a template for telomere extension
- synthesize short bit of DNA that base pairs and blocks telomerase activity via RNA
imetelstat
- administered by IV infusion
- green “tail” is 16 carbon-long lipid to improve movement across the cell membranes to increase potency & improve pharmacokinetic & pharmacodynamic properties (covalent binding)
- membrane is a compartment barrier (to the nucleus)
- imetelstat binds to template region of RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzymatic activity (into nucleus)
- clinical trials results: suppresses proliferation of malignant progenitor cells aiding recovery of normal hematopoiesis in patients with hematologic myeloid malignancies
telomerase function blocked by AZT
- azidothymidine: structure does not allow additional ntds to be added; the consequence of 5’ –> 3’ rule
- at end of the chromosome
*no more 3’ OH because needed so terminated
thymidine vs. zidovudine (AZT)
*different chemical structure
thymidine
- binds to previous ntd (3’ OH group allows binding to next ntd)
- continues nucleic acid chain
- needed in telomere
AZT
- binds previous ntd
- azido (-N3) group in 3’ position (not available for 3’ elongation)
- thymidine analogue
- phosphate group of next ntd cannot bind azido-thymidine; synthesis stops
topoisomerases
*break and restore backbone
- nuclear enzymes supporting DNA replication
- dual functionality via covalently binding to DNA
- induce single-stranded breaks (covalent) –> DNA unwinds helix is relaxed, ssDNA available for replication (DNA polymerase)
- topoisomerase rejoins DNA ends in the relaxed region and dissociates from the relaxed double-stranded region
- double helix not conducive to polymerization
topo
- elevation
- major + minor DNA
- impede DNA replication when in helix
What happens if religation is inhibited?
*drug
- accumulation of single-stranded breaks
- irreversible double-stranded breaks
- leads to cell death
- double-stranded breaks target for degradation
DNA helix as drug target
- camptothecins = natural & synthetic alkaloids (cytotoxic chemotherapy drug) –> insert between DNA base pairs (intercalation because planar)
- partially inhibit topoisomerase 1
- initial cleavage occurs
- drug (yellow) H-bonds to topoisomerase (amino acid interactions) (blue protein ribbon) and intercalates between base pairs
- re-ligation step is inhibited
- single-strand breaks accumulate (to double-strand breaks)
clinical use of camptothecin
- topoisomerase activity is required at beginning of the S phase to relax the helix & allow access for DNA polymerase
- cancer cells must be in the S phase (most activity)
- little effect with slow or non-cycling cells
- treatment duration > cell cycle length
*only active when cells replicate DNA –> does nothing if not replicating
chromatin –> clinical therapies
*chromatin = DNA and associated proteins
- 372 trials completed and 125 in progress for HDACi
- entinostat = specific inhibitor of class 1 HDACs
- HDACs = histone deacetylases
- trials histone enzymes inhibitors
Clinical phases
phase 1: test a new drug in a small group of people, for the first time to evaluate a safe dosage range and side effects.
phase 2: drug or treatment given to a larger group of people to see effectiveness and further evaluate its safety
phase 3: drug is given to large groups to confirm effectiveness, monitor side effects, compare to commonly used treatments and collect information to safety
phase 4: post trial studies for additional information including drugs risk, benefits, and optimal use.
phase designation
*HDACi at all phases of clinical trials
- HDACs modify chromatin protein (phase 2)
- histones, enzymes, inhibitors (phase 4)
gene expression & regulation
gene expression & regulation
- DNA <-> protein interaction affects gene expression (transcription)
- post-translational modifications of chromatin proteins ex. histones, control transcription access to gene
- genome (DNA sequence) and epigenome (chemical modifications to DNA & associated proteins or everything associated) –> phenotypic traits (ex. normal vs. cancer cell growth)
- DNA sequence –> mRNA –> protein –> cell
- non-sequence modifications to DNA & chromatin protein also influence expression (ex. histone acetylation)
- coding information from DNA to mRNA
- transcription initiated at the promoter region
- endogenous and clinically used compounds (like certain drugs & hormones) can affect access to promoter & therefore gene expression (transcription)
*nuclear import/export issues for modifying enzymes & mRNA
chromatin structural elements
- DNA + protein
- histones and nucleosomes
histones
*major protein associated with DNA in the nucleus (group of proteins)
- small (~120aa), basic proteins
- contain numerous lysine
- lysine R group has positively charged NH3+
- interacts with negatively charged DNA phosphate (backbone)
- 5 histone types
- 4 types in the core, 1 as a spacer between nucleosomes
*packing based on charge
nucleosomes
- 4 pairs of histones (pairs) at the core (makeup core of nucleosome)
- ~146bp DNA wraps around in ~1.7 turns
*8 individual histone proteins (octamer)
- tightly wrapped because of + and - charge interactions
- beads are nucleosomes and spacer region is string
chromatin structure
multiple nucleosomes = “beads on a string”
- ~10 nm diameter
- packing depends on histone post-translational modification
chromatin fiber
- ~30 nm diameter
- the basic structure of interphase chromosome
- histone phosphorylation, methylation, & acetylation affect packing ~2 meters DNA in nuclear diameter < or = micron
histone acetylation
- linker histone (5th) and octamer core
*chromatin packing: DNA & protein charges (denser based on + and -)
histone acetylation
- NH3+ group on lysine is covalently modified - reduces interaction between histones and DNA
- histone acetyl transferases (gene expression) - HAT’s transfer acetyl group to lysine side chain
- histone deacetylases (gene silencing) - HDAC’s remove acetyl groups from lysing
- packaging based on + and -
*multiple types of HATs and HDACs within but make contrast
(t/f) histone acetylation is the only PTM
false; can have many but focus on acetylation because important for packaging DNA
histone acetylation reversibility
*modification repeatedly reversible
- chromatin: packed –> unpacked
- transcription: limited –> permitted
- deacetylation via HDACs results in electrostatic binding between DNA and lysine in histone backbone protein (R group) –> gene silencing because packed/dense and not available for transcription (compaction of histone protein and DNA)
- acetylation results in acetylation of lysine which makes it have no electrical charge (covalently attached) and results in no binding and release of DNA –> facilitate gene expression because chromatin open and active/available to transcription factors for gene expression
HDACs
*reversible histone acetylation
- histone deacetylases
- remove acetyl (COCH3) groups
- lysing NH3+ available for interaction with PO4-
- chromatin is compacted
- 20-30nm diameter chromatin “solenoid”
- expression is restricted to completely silenced
- the extension of histone proteins (lysine) is positive
HDAC inhibitors
*gene-level effects
- histone acetyl not removed
- HAT’s continue to acetylate histones
- “open” chromatin structure with more access for RNA pol II
- expression is maintained or initiated from many but not all genes
- transcription of genes encoding cell replication-suppressing proteins is typically increased (stop cancer cells from growing)
- encode growth suppressor proteins
- different amounts of HDACs in different areas
*ex. vorinostat = HDACi related to entinostat
(t/f) transcription is via polymerase II
true; transcribes mRNA
promoters simplified
- compact heterochromatin (silent) - HDAC –> lo/no acetyl groups
- other transcription repressing proteins (PRC1) present
- transcription factors bind upstream of TATA
- HATs & chromatin-modifying enzymes & RNA pol ii recruited (add acetyl groups to open up for transcription)
- upstream promoter (non-coding region) and downstream coding
- attracts proteins
- multiple, sequential RNA pol recruited for subsequent transcription initiation events
- additional chromatin-modifying proteins recruited to allow transcription progression
- downstream histones and coding region
promoters
-specific DNA sequence at start of gene
- RNA pol binding site
- also includes distant sequences further upstream where other regulatory proteins bind
- 100’s to 1000’s of nucleotides long
- TATA box
transcription factors
- aid RNA pol interaction with promoter
- bind to DNA - 100’s to 1000’s of ntds upstream of “TATA” box
- therefore promoters may extend 100’s - 1000’s of ntds upstream
- bind things that facilitate transcription
terminator sequence
cause release of pol downstream gene
proteins with 2 domains
- transcription (other regulatory proteins to bind) - specific factors
- DNA-binding domain (DBD)
- activation domain
DNA-binding domain (DBD)
- protein-DNA physical interaction
- interacts w/ major or minor groove of helix
- DBD alone is not sufficient to stimulate transcription
- particular nucleotide sequence
- several common structures derived from protein’s a-helices
- leucine zipper, helix-loop-helix, zinc finger motifs
- ex. leucine (inner red residues) zipper model for AP-1 (fos-jun)
activation domain
- stimulates transcription if contiguous with the DNA-binding domain
- scaffold to recruit more transcription-enhancing proteins
- often rich in aspartate & glutamate residues (acidic aa may interact with basic histone protein)
- interacts with the activation domain of another protein (synergy)
mRNA processing
*mRNA’s are processed before leaving the nucleus (ii)
- introns = interrupting the non-coding sequence
- present in most newly transcribed eukaryotic mRNAs
- excised from mRNA by spliceosomes (proteins & short RNA’s form scaffold for cleavage, excision, and reforming bond
- exons = expressed coding sequences
- covalent bonds re-established after excision to link together
- export from nucleus after splicing, capping & tailing is complete
- introns and exons from DNA template
mRNA and splicesome structure
mRNA
- partially processed = 5’ cap, poly-A tail, exons, and introns
- ligation to fully processed with only exon
spliceosome
- enzymatically active
- breakage of intron looped out
- temporarily base pair to intron to cleave
mRNA processing overview
- both ends modified may slow degradation & increase mRNA half-life
- modified nucleotide (guanine) “cap” added to 5’ end
- poly-A tail added to 3’ end (post-transcription no template)
- cap & poly-A tail are not gene-encoded
- additions are post-transcriptional & DNA template independent
mRNA sequence
*mRNA content (final form as exported from nucleus)
- mRNA is no longer than amino acid coding info it contains
- leader & trailer are transcribed from DNA
- leader: responsible for mRNA interaction with ribosome subunit
- trailer: AAUAAA signals transcription end & addition of poly A (variable tail length)
- start codon = methionine
shane burcaw
- spinal muscular atrophy
- takes spinraza
spinal muscular atrophy (mRNA processing in disease)
- ~35% of human genetic diseases are associated with splicing
- SMA = leading genetic cause of mortality in infants <2 yo
- insufficient muscle innervation –> paralysis & death (issues making SMN proteins)
- shane’s diagnosis
SMN protein (survival motor neuron)
required for nerve-muscle innervation
- protein from either SMN1 or SMN2 gene suffices
SMN1 genes
often 1 allele is deleted & the other mutated. Truncated protein from a mutated gene is degraded –> degeneration of spinal cord motor neurons
SMN2 genes
sequence normal but SMN2 pre-RNA variable spliced –> splicing usually removes needed #7 exon resulting protein is easily degraded
- cannot compensate for SMN1 protein loss
*cannot change SMN1
guided mRNA processing disease treatment
drug screening
- ~558,000 compounds assayed in cells in Petri dishes 17 increased transcription or promoted RNA splicing
- good news/initial bad news/recent good news: improve cell survival but effects not all specific to SMN mRNA
alternatively how-to specifically target SMN2 pre-RNA
- take advantage of the defined SMN2 splicing process that is snRNP guided to splice introns flanking exon 7 of pre-mRNA, thus retaining exon 7
mRNA disease treatment
*shorter SMN2 protein degraded
1) chemical approach
2) targeted approach
*goal to retain exon 7
mRNA processing: a “druggable” target
*This is a small molecule drug-chemical approach
- additional chemical screening yielded a candidate oral drug (risdiplam) that targets SMN2 pre-RNA splicing but also some other pre-mRNA transcripts
- FDA approved becoming the third disease-modifying treatment for SMA
mRNA splicing: a therapeutic target
- the mechanism by which ASO (anti-sense oligonucleotide) stimulates SMN2 exon 7 inclusion appears to be complex
- normally shortened SMN2 protein degraded
- spinraza = injection of synthetic DNA in neurons innervating muscle cells –> as oligo serves as splicing guide to full-length SMN2 protein stable (takes place of missing SMN1 protein)
tRNA structure
- tRNA - one for each amino acid
- no tRNA are the same because of different nucleotide sequences and base pairing (changes 3D structure)
- anti-codon base-pairs with codon on mRNA
- intramolecular base pairing (rungs on ladder –> 3D consequence)
- carries amino acid via ester bond at 3’ terminus (charged)
- intramolecular base-pairing responsible for 3D structure (vs. flattened “cloverleaf”)
- anticodon loop available based on intramolecular base pairing region
- different enzyme complexes adding amino acids too
tRNA function
- mRNA translation into protein
- tRNA “reads” codon of mRNA
- mRNA codons do not interact directly with aa
tRNA activation
- requires amino acid, tRNA, enzyme & energy source
- aminoacyl-tRNA synthetase (enzyme) unique and specific enzyme complex
- uniquely match to tRNA elbow and amino acid (lock + key = dimensional)
*selectivity
tRNA activation steps
- correct amino acid is bound to tRNA by enzyme complex
- one for each tRNA-amino acid combination using ATP
- transiently adds ATP to convert to AMP (energy dependent)
- AMP is exchanged for last nucleotide of tRNA
- AMP is released; amino acid covalently linked to tRNA via 3’-OH (exposed)
- selectively occurring
- activated tRNA is released & ready for codon recognition
*activated amino acid = aminoacyl tRNA = charged tRNA
interpreting codons
- example of codon redundancy = stop codons, LEU, VAL, ARG
- redundancy = different codon sequence bring in same amino acid
- examples of uniquely encoded amino acids = MET, TRP
stop codons
- UAA, UGA, UAG
- stop translation
- different sequences do the same thing = redundant
Consequence of a sequence variant changing a codon from CGU to CGG?
- no change in protein sequence
- redundant
start codon
*AUG - start - MET
- nonredundant codon
- first codon - always at the start
ribosome structure
- general features: 1 large & 1 small subunit
- each has characteristic proteins & rRNAs
- rRNAs have a secondary structure from intramolecular base pairing similar to tRNA
- rRNAs have catalytic activity (based on 3D conformation from intramolecular base pairing) for peptide bond formation –> covalent bonds along protein backbone
- flatten out and see intramolecular forces (long)
large vs. small subunit
- both made of RNA and protein
- proteins intertwine with different sizes of rRNA
- transcription of rRNA and subunit assembly occurring in the nucleolus
- not just crammed together - specific shape dependent on intramolecular base pairing
- small = rRNA 16S and rProtein
- large = rRNA 5S and 23S and rProtein
*sedimentation larger is slower
eukaryotic vs. prokaryotic ribosomes
*mammalian vs. bacterial
- size: eukaryotic (80S) vs. bacterial (70S), svedberg units (large & small subunits) slower in centrifuge –> larger
- sequence: different protein and rRNA in eukaryotic & prokaryotic ribosomes (slightly different structure/size but both translate to protein)
- surfaces: some 3D differences from rRNA & rProtein
- antibiotic interaction: rRNA & rProtein spacing in mammalian ribosomes is typically too small to allow efficient entry of antibiotics (difference)
antibiotics (and ribosome)
- different antibiotics target different steps of translation
- differently, interact with different regions of the ribosome
- tetracycline: blocks tRNA interaction with the ribosome
- erythromycin: blocks ribosome moving along mRNA
- streptomycin: blocks interaction of tRNA & mRNA codon
Is there an advantage to the combined use of antibiotics?
- no treatment is 100% effective –> one may fail
- a combination that targets distinctly different steps
- combination therapy
- compensate for the inefficiency of one
three stages of translation
initiation, elongation, termination
Initiation - translation
- mRNA initiation sequence binds to mRNA to small ribosome subunit
- includes kozak consensus sequence around start codon (commonly found and small subunit recognizes)
- methionine-tRNA binds to start codon (AUG = MET) via anticodon of tRNA - next to initiation sequence
- large subunit binds to small subunit: MET tRNA fits in the P site of the large subunit
*mRNA has 5’ cap and poly-A tail because it is processed RNA - to increase stability and t1/2
kozak consensus sequence
- AUG 100% match always present
- larger letter = more often found
- whole sequence is very commonly occurring in mRNA
initiation sequence onward - initiation
- initiation sequence = short, non-coding that allows for the initiation of small subunit association
- move along until initiator tRNA reaches AUG
- aminoacylated tRNA complex attracts large ribosomal subunits and binds in p site
- 3 clefts/parking spots
- e = exit, p = peptide, a = activated aa (where the second tRNA will go with amino acid)
elongation - translation
*requires GTP hydrolysis
- ribosome catalyzes the formation of peptide bond between amino group of incoming aa & carboxy terminus of growing peptide (covalent bond)
- ribosome advances one codon
- broken polypeptide chain in p site transferred to amino acid in a site
- all sites move over
termination - translation
- requires release factors
- elongates until stop codon (UAA, UAG or UGA) at a site - attract release factor redundant
- as long as there are more codons there is elongation
- finished polypeptide released by hydrolysis from last tRNA
- ribosome splits into subunits (disassembly)
- available for binding to initiation sequence on another mRNA
recycling of mRNA
- protective caps –> can still continue and restart with initiation (reused)
- if caps are hydrolyzed –> mRNA breaks down and is unavailable for translation
(t/f) tRNA recognizes stop codon
false; release factor
signal peptide
- aka leader peptide, signal sequence
- signal peptide held in membrane - rest of protein goes into ER lumen
- signal peptidase in rER lumen cleaves off protein
- new protein released to ER lumen
- signal peptide released to cytoplasm % degraded
secreted vs. cytosolic proteins
- where in or outside cell synthesized protein ends up
- all translation initiates on free ribosomes in the cytoplasm
- some is completed in the cytoplasm
- but proteins with signal peptide @ N-term direct ribosome, mRNA & protein to ER
secreted vs. cytosolic proteins process
*common pool of ribosomal subunits in cytosol
no signal peptide at N-term
- signal sequence initiates translation
- many ribosomes translate newly made protein
- no directions so start and terminate in cytoplasm after release factor
red N-term = aa of signal peptide
- translation starts in cytoplasm
- 5’ end codes for amino acids for signal peptide
- recognize complex on ER –> direct protein to surface of ER membrane
- makes rough ER by bringing ribosomal complex
- once N-terminus is exposed, it is directed to ER
how some ER becomes rough
- association of protein with pore complex on ER
- signal peptidase cleaves protein which is released into ER lumen (first step of ER process) - proteolytic cleavage
- cleavage generates new N-term for protein without initiator MET
- mature polypeptide chain with new N-term delivered into ER lumen (protein product funneled in)
- signal peptide released from ER membrane & degraded (reuse amino acids)
- the protein in ER gets further processing, delivery to other organelles, or secretion
secreted proteins - insulin example
i*into body and bind with receptor on cell
- rER lumen: preproinsulin, signal seq cleaved –> proinsulin
- golgi lumen: proinsulin, 2 additional cuts by enzyme –> C chain released
- golgi lumen: A & B chains linked by 2 disulfide bonds –> mature insulin; example of organelle compartment specialization
- secretory vesicles bud off golgi
- fuse to cell membrane upon cell’s receipt of secretion stimulus
- deliver contents in pancreas to pick up by bloodstream (pancreatic cell)
insulin process
- insulin translated with signal peptide
- signal sequence guides to rER lumen
- transported between organelles via vesicles and fuse
- golgi is more post translational processing and cleavage
- secretory vesicles are good examples of proteins with signal peptides
- insulin = fully processed
cytosolic proteins - translation
- cytosolic polypeptides lack signal sequence
- synthesized by ribosomes that stay free in cytoplasm
- nascent protein folding and chaperones
- initiation + completion on ribosome
- heat shock proteins (chaperones) transiently bind for protein-protein interactions
- dissociation of HSP after folded
nascent protein folding and chaperones
- milliseconds to seconds - protein compaction: for the secondary structure to shield hydrophobic groups from aqueous cytoplasm
- seconds to minutes - chaperone interaction: for many, not necessarily all new cytoplasmic proteins HSP: heat shock protein, chaperone example
examples of cytosolic proteins
any cytoskeletal protein, DNA, RNA polymerases, histones
duchenne muscular dystrophy
*dystrophin mutation
- mRNA amino acid codon mutated to premature termination/STOP codon (PTC)
- truncated protein degraded (short)
- disorganized cytoskeleton; poor muscle function
- normally orders muscle but no protein present in disease state
Duchenne muscular dystrophy PTC
- termination/release factors at PTC
- can eliminate continued protein coding sequence
- force ribosome over codon
ataluren
*therapy for diagnosis of duchenne muscular dystrophy
- interacts with ribosome
- facilitates 1/3 or 2/3 match to anti-codon of amino acid encoding tRNA
- III = stop codon binding release factor
- IIX = partial: UAA (STP) read as UAC (TYR)
- allows read-through of premature stop codon within mRNA but not as usual 3’ position
- translation bypass mutated codon; restores production of full-length protein (elongation continues)
- change ribosome conformation - termination factor no longer fits in a site but attracts a different amino acid
polypeptides - degrees of structural organizaiton
- primary = linear aa sequence
- secondary = helical or sheet formation within protein
- tertiary = overall protein shape
- quaternary = > or = 2 separate proteins forming complex
- ex. hemoglobin quaternary structure - two a chains and two b chains + 4 heme groups
- structural importance = enzyme active sites, receptor ligand binding, ab recognition of antigen, Na+/K+ pump
covalent modifications of polypeptides
- hydroxylation: collagen proline (to hydroxyproline) improves stability
- phosphorylation: IF serine or threonine, causes disassembly
- phosphorylation: Na+/K+ pump, affects ion transport
- acetylation
