DNA Replication, Gene Expression & Protein Synthesis Flashcards
DNA structure
3D structure, 2 helices wrapping around each other, surface as major (wide spiral) and minor grooves (narrow spiral), size of groove affects access to bases within helix
DNA and drug pharmacology
target for chemotherapy drugs through various types of interactions, strand breaker, non-covalent interactions: not chemically interacting but fitting, covalent complexes: cis platin - platinum-based
Non-covalent interactions with DNA
intercalation between bases - fitting into (in space/opening), minor grooves, major grooves, or spanning both
Cis platin
anticancer chemotherapy drug, covalent binding - specific to 1 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 1 guanine nucleotide, interstrand - consecutive nucleotides opposite strand = about 3-5% (one strand across another), intrastrand - consecutive nucleotides, about 80-90% (2 covalent interactions on one strand - same backbone), intrastrand - nearby nucleotides, about 3-5% (skip residue), either strand - guanine and protein, about 3%
How and 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 template for polymerase, slower growing normal cells less likely affected than faster replicating/metabolizing cancer cells, chemotherapy has a negative impact on cancer cells, nonselective so not only cancer cells impacted (all cells that replicate/transcribe DNA)
DNA replication overview
replication events/requirements, chromosome ends (5’ to 3’ rule) and unwinding (topoisomerase), possible targets for chemotherapy, topoisomerase unwinds double strand DNA, 5’ to 3’ rule because added onto exposed 3’ hydroxyl, double helix slows down DNA polymerase so needs to be unwound by topoisomerase
the 5’ to 3’ rule
templates are anti-parallel, RNA primer required to provide initial 3’ OH for DNA nucleotides added on replication proceeds 5’ to 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 lagging strands
leading strand - continuous synthesis (faster), one RNA primer and synthesis towards replication fork: lagging strand - short and discontinuous synthesis yields separate lengths of DNA (Okazaki fragments) so slower, RNA primers removed by RNAseH 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’ to 3’ rule
leading and lagging strand, chromosome ends (telomeres) shorten with repeated round 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
What are the consequences of repeated rounds of DNA replication?
shortened each time cell replicates DNA, both ends of chromosome (telomere) are shortened
Chromosome ends
telomeres = ends of linear eukaryotic chromosomes, protect and stabilize internal section of chromosome, conserved, short, highly repeated sequence, about 6-9 nucleotide (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 replacing (loses ability to replicate DNA), 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 problems’ of DNA synthesis
DNA polymerases need RNA primer to start replication, 20 to 200 single strand end left after removal of terminal primer, single strand region removed by exonuclease and 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 nucleotides 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 and 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
majority of normal cells do not produce telomerase (somatic cells), consequences for tissue/organ, telomeres shorten then cell stops dividing then replicative senescence, accumulated tissue “wear and 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, and gastrointestinal cells: very low levels, tissues where replacement and renewal is critical, some shortening still crus because level is too low, crypt is location for high replication (more telomerase) and lower levels of telomerase, at the time the cells are shed and replaced by replicative cells at the crypt (travel up), skin cells have a low level of replacement = low telomerase
Cancer cells and telomerase
about 90% of cancer types have high telomerase levels, telomerase levels increases from early to late stage cancer, telomeres maintained and cells divide and escape replicative senescence (no internal clock), the other about 10% of cancer cells maintain telomere ends, alternative lengthening of telomeres = sister chromosome serves as template in process similar to homologous recombination, extended telomere of matched chromosome
DNA replication as drug target
telomerase function blocked by “antisense” DNA, telomerase function blocked by AZT
Telomerase function blocked by “antisense” DNA
restore clock to stop replication, complementary to DNA component of enzyme (plug up to preoccupied with something else), inactive telomerase complex (DNA+RNA+protein), RNA not available as template for telomere extension, synthesize short bit of DNA that base pairs and blocks telomerase activity with RNA
Imetelstat
administered by IV infusion, green “tail” is 16 carbon-long lipid to improve movement across cell membrane to increase potency and improve pharmacokinetic and pharmacodynamic properties (covalent binding), membrane is compartment barrier (to nucleus), imetelstat binds to template region of RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzyme activity (into nucleus), clinical trials results: suppresses proliferation of malignant progenitor cells aiding recovery of normal hematopoiesis in patient with hematologic myeloid malignancies
Telomerase function blocked by AZT
azidothymidine: structure does not allow additional nucleotide to be added which is consequence of 5’ to 3’ rule, at end of chromosome, no more 3’ OH because needed so terminated
Thymidine vs. zidovudine (AZT)
different chemical structure, thymidine - binds to previous nucleotide (3’ OH group allows binding to next nucleotide), continues nucleic acid chain, needed in telomere; AZT - binds previous nucleotides, azido (-N3) group in 3’ position (not available for 3’ elongation), thymidine analogue, phosphate group of next nucleotide cannot binds azido-thymidine; synthesis stops
Topoisomerases
break and restore backbone, nuclear enzymes supporting DNA replication, dual functionality with covalently binding to DNA, induce single-stranded breaks (covalent) then DNA unwinds helix is relaxed so ssDNA available for replication (DNA polymerase), topoisomerase rejoins DNA ends in relaxed region dissociates from relaxed double-stranded region, double helix not conductive to polymerization
Topo
elevation, major and minor DNA, impede DNA replication with in helix
What happens if religation is inhibited?
accumulation of single-stranded breaks, irreversible double-stranded breaks, leads to cell death
Drug helix as drug target
Camptothecins = natural and synthetic alkaloids (cytotoxic chemotherapy drug) - insert between DNA base pairs (intercalation because planar), partially inhibit topoisomerase 1, initial cleavage occurs, drug hydrogen bonds to topoisomerase and intercalates between base pairs, religation step is inhibited, single-stranded breaks accumulate (to double strand breaks)
Clinical use of camptothecin
topoisomerase activity required at beginning of S phase to relax helix and allow access for DNA polymerase, cancer cells must be in S phase (most activity), little effect with slow or non cycling cells, treatment duration depends on cell cycle length, only active when cells replicate DNA
Chromatin - clinical therapies
chromatin = DNA and associated proteins, entinostat = specific inhibitor of class 1 HDACs, HDACs = histone deacetylases, trials histone enzymes inhibitors
Phase 1 clinical trials
test a new drug in a small group of people (10-80) for the first time to evaluate a safe dosage range and side effects
Phase 2 clinical trials
drug or treatment is given to a larger groups of people (100-300) to see if it is effective and to further evaluate its safety, HDACs modify chromatin protein
Phase 3 clinical trials
drug is given to large groups (1,000-3,000) to confirm effectiveness, monitor side effects, compare to commonly used treatments, and collect information on safety, HDACi at all phases
Phase 4 clinical trials
post trail studies for additional information including drug’s risks, benefits, and optimal use, histones enzymes inhibitors
Gene expression and regulation
DNA to and from protein interaction affects gene expression (post transitional modifications of chromatin protein, histones, control transcriptional access to gene), genome (DNA sequence) and epigenome (chemical modification to DNA and associated proteins) to phenotypic traits, coding information from DNA to mRNA, nuclear import/export issues for modifying enzymes and mRNA
Coding information from DNA to mRNA
transcription initiated at promoter region, endogenous and clinically used compounds (like certain drugs and hormone s) can affect access to promoter and therefore gene expression (transcription)
Sequence of gene expression and regulation
DNA sequence to mRNA to protein to cell, non-sequence modification to DNA and chromatin protein also influence expression (histone acetylation)
Chromatin structure DNA and protein
histones: small (about 120 amino acids) and basic proteins, contain numerous lysines, lysine R group has positively charged DNA phosphate, 5 histone types which are 4 types in core and 1 as spacer between nucleosomes; nucleosomes: 4 pairs of histones at core, about 146 base pairs DNA wrap around in about 1.7 turns
Chromatin structure
multiple nucleosomes = “beads on a string”, about 10nm diameter, packing depends on histone post-translational modifications; chromatin fiber - about 30nm diameter, basic structure of interpose chromosome, histone phosphorylation, methylation and acetylation affect packing so about 2 meters DNA in nuclear diameter
Histone acetylation
chromatin packing: DNA and protein charges, NH3+ group on lysine is covalently modified - reduces interaction between histones and DNA, Histone acetyl transferases (gene expression) - HATs transfer acetyl transfer acetyl groups to lysine side chain, histone deacetylases (gene silencing) - HDACs remove acetyl groups from lysine, multiple types of HATs and HDACs, modifications repeating reversible
HDACs
histone deacetylases, remove acetyl (COCH3) groups, lysine NH3+ available for interaction PO4-, chromatin is compacted, 20 to 30nm diameter chromatin “solenoid”, expression is restricted to completely silenced
HDAC inhibitors
gene-level effects - histone acetyl not removed, HATs continue to acetylate histones, “open” chromatin structure with more access for RNA polymerase 2, expression is maintained or initiated from many but not all genes, transcription of genes encoding cell replication-suppressing proteins is typically increased
Promoters steps
compact heterochromatin (silent) - HDAC so low or no acetyl groups and other transcription repressing proteins (PRC1) present, transcription factors bind upstream of TATA (gene), HATs & chromatin-modifying enzymes & RNA polymerase 2 recruited (add acetyl groups to open up for transcription), upstream promoter (non-coding region) and downstream coding and attracts proteins, multiple, sequential RNA pol recruited for subsequent transcription initiation events, additional chromatin-modifying proteins recruited to allow transcription progression
Promoters
specific DNA sequence at start of gene, RNA polymerase binding site, also includes distant sequences further upstream where other regulatory proteins binds, 100s to 1000s of nucleotides long, TATA box
Transcription factors
aid RNA polymerase interaction with promoter, binds to DNA - 100s to 1000s of nucleotides upstream of “TATA” box, therefore promoters may extend 100s, 1000s of nucleotides upstream, binds things that facilitate transcription
Terminator sequence
cause release of polymerase downstream gene
Proteins with 2 domains
transcription (other regulatory proteins to bind) - specific factors, DNA-binding domain, activation domain
DNA-binding domain
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
Activation domain
stimulates transcription if contiguous with DNA-binding domain, scaffold to recruit more transcription-enhancing proteins, often rich in aspartate & glutamate residues (acidic amino acid may interact with basic histone protein), interacts with activation domain of another protein (synergy)
mRNA processing
mRNAs are processed before leaving nucleus, introns = interrupting 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 spliceosome 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
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 information 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
spinal muscular atrophy (mRNA processing in disease)
about 35% of human genetic diseases associated with splicing, spinal muscular atrophy = leading genetic cause of mortality in infants less than 2 years old, insufficient muscle innervation leads to paralysis & death (issues making SMN proteins)
Protein and gene information form spinal muscular atrophy
SMN protein (survival motor neuron) required for nerve-muscle innervation, protein from either SMN1 or SMN2 gene suffices, SMN1 genes (often 1 allele deleted & the other mutated) truncated protein from mutated gene is degraded leads to degeneration of spinal cord motor neurons, SMN2 gene sequence normal but SMN2 pre-RNA variable spliced leads to 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 - about 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 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 spinal muscular atrophy
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 so as oligo serves as splicing guide to full-length SMN2 protein stable (takes place of missing SMN1 protein)
Gene expression - review
DNA gene to mRNA transcript to function or structural protein to cell activity or structure to organ activity/structure to organism, transcription of DNA in nucleus into RNA, rRNA ribosomal RNA - polymerase I, mRNA messenger RNA - polymerase II, tRNA transfer RNA - polymerase III
RNA polyermase 2
reads 3 nucleotides per codon, with 4 nucleotides available, more than enough to cover the 20 encoded amino acids, DNA gene to mRNA transcript to protein, consequences of mutations
Mutations - review
mutations change coding sequence, may occur in somatic or reproductive cells, can occur spontaneously (DNA polymerase error) or by radiation or chemical exposure
Nucleotide substitution
may result in replacement of one amino acid with another, example - sickle cell anemia allele single base change in DNA results in aa change in hemoglobin, or no change if new codon codes for same aa (coding redundancy)
Insertion or deletion of bases
results in phase shift of three base reading frame, affects all codons after mutation, results in different amino acid sequences, almost always results in non-functional polypeptide
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 complex adding amino acids too
tRNA function
mRNA translation into protein, tRNA “reads” codon of mRNA, mRNA codons do not interact directly with amino acid
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)
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
Start codon
*AUG - start - MET - nonredundant codon, first codon - always at start
Ribosome structure
general features: 1 large & 1 small subunit, each has characteristic proteins & rRNAs, rRNAs have 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 leads to 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 ribosome typically too small to allow efficient entry of antibiotic (difference)
Antibiotics (and ribosomes)
different antibiotics target different steps of translation, differently interact with different regions of ribosome
- tetracycline: blocks tRNA interaction with ribosome
- erythromycin: blocks ribosome moving along mRNA
- streptomycin: blocks interaction of tRNA & mRNA codon
Is there an advantage from combined used of antibiotics?
no treatment is 100% effective so one may fail, combination that targets distinctly different steps, combination therapy, compensate for inefficiency of one
Stages of translation
initiation, elongation, termination
Initiation
- 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 P site of 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 sequence = short, non-coding that allows for initiation of small subunit association, move along until initiator tRNA reaches AUG, aminoacylated tRNA complex attracts large ribosomal subunit and binds in p site
- 3 clefts/parking spots
- e = exit, p = peptide, a = activated amino acid (where second tRNA will go with amino acid)
Elongation
requires GTP hydrolysis, ribosome catalyzes 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
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 so can still continue and restart with initiation (reused), if caps are hydrolyzed then mRNA breaks down and unavailable for translation
Signal peptide
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 cytoplasm, some is completed in cytoplasm, but proteins with signal peptide at N terminus direct ribosome, mRNA & protein to ER
Secreted vs cytosolic proteins process
common pool of ribosomal subunits in cytosol
no signal peptide at N-terminus, signal sequence initiates translation, many ribosomes translate newly made protein, no directions so start and terminate in cytoplasm after release factor; red N-terminus = amino acid of signal peptide - translation starts in cytoplasm, 5’ end codes for amino acids for signal peptide, recognize complex on ER then 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), protein in ER gets further processing, delivery to other organelles, or secretion
Secreted proteins - insulin example
into body and bind with receptor on cell
rER lumen: preproinsulin, signal seq cleaved which is proinsulin, golgi lumen: proinsulin, 2 additional cuts by enzyme so C chain released, golgi lumen: A & B chains linked by 2 disulfide bonds then 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 secondary structure to shield hydrophobic groups from aqueous cytoplasm, seconds to minutes - chaperone interaction: for many, not necessarily all new cytoplasmic proteins 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 to continue 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 organization
- primary = linear amino acid sequence
- secondary = helical or sheet formation within protein
- tertiary = overall protein shape
- quaternary = more or equal to 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 modification or polypeptides
- hydroxylation: collagen proline (to hydroxyproline) improves stability
- phosphorylation: IF serine or threonine, causes disassembly
- phosphorylation: Na+/K+ pump, affects ion transport
- acetylation
