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DNA, Gene expression and protein synthesis Flashcards

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1
Q

DNA structure

A
  • 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
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2
Q

DNA & drug pharmacology

A
  • 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
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3
Q

non-covalent interactions with DNA

A
  • 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

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4
Q

cis platin

A

*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
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5
Q

types of covalent cis platin binding

A

*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%

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6
Q

How & why is cis-platin binding detrimental to cancer cell? Are only cancer cells affected?

A
  • 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)

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7
Q

DNA replication overview

A
  • 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
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8
Q

the 5’ –> 3’ rule

A
  • 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
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9
Q

2 kinds of daughter strands

A

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
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10
Q

ligase

A

*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
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11
Q

a consequence of the 5’ –> 3’ rule

A

*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
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12
Q

What are the consequences of repeated rounds of DNA replication?

A

*shortened each time cell replicates DNA

  • both ends of the chromosome (telomere) are shortened
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13
Q

chromosome ends

A
  • 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

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14
Q

telomere shortened too much

A
  • 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.
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15
Q

end replication problem’ of DNA synthesis

A
  • 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
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16
Q

how to rebuild telomeres

A

*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

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17
Q

telomerase structure

A
  • 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
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18
Q

telomerase in normal cells

A

*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
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19
Q

telomerase activity

A
  • 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
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20
Q

cancer cells and telomerase

A
  • ~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
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21
Q

(t/f) telomerase & alternative processes may be anticancer targets to block chromosome maintenance

A

true

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22
Q

DNA replication as a drug target

A
  • telomerase function blocked by “antisense” DNA
  • telomerase function blocked by AZT
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23
Q

telomerase function blocked by “antisense” DNA

A
  • 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
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24
Q

imetelstat

A
  • 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
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25
Q

telomerase function blocked by AZT

A
  • 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

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26
Q

thymidine vs. zidovudine (AZT)

A

*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
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27
Q

topoisomerases

A

*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
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28
Q

topo

A
  • elevation
  • major + minor DNA
  • impede DNA replication when in helix
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29
Q

What happens if religation is inhibited?

A

*drug

  • accumulation of single-stranded breaks
  • irreversible double-stranded breaks
  • leads to cell death
  • double-stranded breaks target for degradation
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30
Q

DNA helix as drug target

A
  • 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)
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31
Q

clinical use of camptothecin

A
  • 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

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32
Q

chromatin –> clinical therapies

A

*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
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33
Q

Clinical phases

A

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.

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34
Q

phase designation

A

*HDACi at all phases of clinical trials

  • HDACs modify chromatin protein (phase 2)
  • histones, enzymes, inhibitors (phase 4)

gene expression & regulation

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35
Q

gene expression & regulation

A
  • 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

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36
Q

chromatin structural elements

A
  • DNA + protein
  • histones and nucleosomes
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37
Q

histones

A

*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

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38
Q

nucleosomes

A
  • 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
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39
Q

chromatin structure

A

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 -)

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40
Q

histone acetylation

A
  • 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

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41
Q

(t/f) histone acetylation is the only PTM

A

false; can have many but focus on acetylation because important for packaging DNA

42
Q

histone acetylation reversibility

A

*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
43
Q

HDACs

A

*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
44
Q

HDAC inhibitors

A

*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

45
Q

(t/f) transcription is via polymerase II

A

true; transcribes mRNA

46
Q

promoters simplified

A
  1. compact heterochromatin (silent) - HDAC –> lo/no acetyl groups
  • other transcription repressing proteins (PRC1) present
  1. 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
  1. multiple, sequential RNA pol recruited for subsequent transcription initiation events
  • additional chromatin-modifying proteins recruited to allow transcription progression
  • downstream histones and coding region
47
Q

promoters

A

-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
48
Q

transcription factors

A
  • 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
49
Q

terminator sequence

A

cause release of pol downstream gene

50
Q

proteins with 2 domains

A
  • transcription (other regulatory proteins to bind) - specific factors
  • DNA-binding domain (DBD)
  • activation domain
51
Q

DNA-binding domain (DBD)

A
  • 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)
52
Q

activation domain

A
  • 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)
53
Q

mRNA processing

A

*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
54
Q

mRNA and splicesome structure

A

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
55
Q

mRNA processing overview

A
  • 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
56
Q

mRNA sequence

A

*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
57
Q

shane burcaw

A
  • spinal muscular atrophy
  • takes spinraza
58
Q

spinal muscular atrophy (mRNA processing in disease)

A
  • ~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
59
Q

SMN protein (survival motor neuron)

A

required for nerve-muscle innervation

  • protein from either SMN1 or SMN2 gene suffices
60
Q

SMN1 genes

A

often 1 allele is deleted & the other mutated. Truncated protein from a mutated gene is degraded –> degeneration of spinal cord motor neurons

61
Q

SMN2 genes

A

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

62
Q

guided mRNA processing disease treatment

A

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
63
Q

mRNA disease treatment

A

*shorter SMN2 protein degraded

1) chemical approach

2) targeted approach

*goal to retain exon 7

64
Q

mRNA processing: a “druggable” target

A

*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
65
Q

mRNA splicing: a therapeutic target

A
  • 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)
66
Q

tRNA structure

A
  • 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
67
Q

tRNA function

A
  • mRNA translation into protein
  • tRNA “reads” codon of mRNA
  • mRNA codons do not interact directly with aa
68
Q

tRNA activation

A
  • 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

69
Q

tRNA activation steps

A
  1. 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
  1. 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

70
Q

interpreting codons

A
  • 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
71
Q

stop codons

A
  • UAA, UGA, UAG
  • stop translation
  • different sequences do the same thing = redundant
72
Q

Consequence of a sequence variant changing a codon from CGU to CGG?

A
  • no change in protein sequence
  • redundant
73
Q

start codon

A

*AUG - start - MET

  • nonredundant codon
  • first codon - always at the start
74
Q

ribosome structure

A
  • 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)
75
Q

large vs. small subunit

A
  • 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

76
Q

eukaryotic vs. prokaryotic ribosomes

A

*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)
77
Q

antibiotics (and ribosome)

A
  • 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
78
Q

Is there an advantage to the combined use of antibiotics?

A
  • no treatment is 100% effective –> one may fail
  • a combination that targets distinctly different steps
  • combination therapy
  • compensate for the inefficiency of one
79
Q

three stages of translation

A

initiation, elongation, termination

80
Q

Initiation - translation

A
  • 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

81
Q

kozak consensus sequence

A
  • AUG 100% match always present
  • larger letter = more often found
  • whole sequence is very commonly occurring in mRNA
82
Q

initiation sequence onward - initiation

A
  • 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)
83
Q

elongation - translation

A

*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
84
Q

termination - translation

A
  • 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
85
Q

recycling of mRNA

A
  • protective caps –> can still continue and restart with initiation (reused)
  • if caps are hydrolyzed –> mRNA breaks down and is unavailable for translation
86
Q

(t/f) tRNA recognizes stop codon

A

false; release factor

87
Q

signal peptide

A
  • 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
88
Q

secreted vs. cytosolic proteins

A
  • 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
89
Q

secreted vs. cytosolic proteins process

A

*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
90
Q

how some ER becomes rough

A
  • 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
91
Q

secreted proteins - insulin example

A

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)
92
Q

insulin process

A
  • 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
93
Q

cytosolic proteins - translation

A
  • 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
94
Q

nascent protein folding and chaperones

A
  • 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
95
Q

examples of cytosolic proteins

A

any cytoskeletal protein, DNA, RNA polymerases, histones

96
Q

duchenne muscular dystrophy

A

*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
97
Q

Duchenne muscular dystrophy PTC

A
  • termination/release factors at PTC
  • can eliminate continued protein coding sequence
  • force ribosome over codon
98
Q

ataluren

A

*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
99
Q

polypeptides - degrees of structural organizaiton

A
  • 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
100
Q

covalent modifications of polypeptides

A
  • hydroxylation: collagen proline (to hydroxyproline) improves stability
  • phosphorylation: IF serine or threonine, causes disassembly
  • phosphorylation: Na+/K+ pump, affects ion transport
  • acetylation

cell bio (4 decks)

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