Molecular Genetics BIOL 5220 Exam 4

Codon

sequence of three adjacent nucleotides that codes for a specific amino acid

transfer RNA (tRNA)

class of RNA molecules, each of which combines covalently with a specific amino acid for use in protein synthesis

aminoacyl-tRNA

tRNA that is charged with an amino acid

aminoacyl-tRNA synthetases

enzymes that catalyze synthesis of an aminoacyl-tRNA at the expense of ATP energy

class 1 aminoacyl-tRNA synthetases

catalytic domain contains a rossman fold, amino acid will bind to the 2’ OH first the be transferred to the 3’ OH

class 2 aminoacyl-tRNA synthetases

catalytic domain has unique alpha-beta fold, amino acid directly binds to the 3’ OH

anticodon

a sequence of three nucleotides on tRNA that pairs with the corresponding codon on mRNA during translation

reading frame

contiguous, non-overlapping set of three-nucleotide codons in DNA or RNA

open reading frame (ORF)

group of contiguous non-overlapping nucleotide codons in a DNA or RNA molecule that does not include a termination codon

nonsense mutation

mutation that results in the premature termination of a polypeptide chain

suppressor tRNA

mutant tRNA that binds to a termination codon but carries an amino acyl residue that can be incorporated into a growing amino acid chain, suppressing the termination signal

nonsense mutation suppression

wild-type mRNA encodes a full-length protein, with CAG encoding the glutamine (Gln). a nonsense mutation changes CAG to UAG, resulting in premature termination. suppressor tyrosine (Tyr) tRNAs can insert an amino acid at this site, allowing translation to continue

rodin-ohno hypothesis

theory that suggests that the two major classes of aminoacyl-tRNA synthetases have evolved from opposite strands of a single ancestral gene, the two classes are reverse compliments of each other; proposes how tRNA aminoacyl synthesis happened

t-box riboswitch

mechanism that regulates the expression of amino acid-related genes; if tRNA is aminoacylated, the riboswitch assumes a structure that allows transcription, while uncharged tRNA leads to a different structure that inhibits transcription

4 primary components of translation

ribosomes, mRNA (template), tRNAs (adaptors that bridge genetic code and amino acids), aminoacyl-tRNA synthetases

ribosomal RNA

RNA molecules serving as components of ribosomes, consistent highly conserved core across bacteria, archaea, and eukaryotes

ribosomal subunits

eukaryotic - 60S and 40S

prokaryotic - 50S and 30S

activation of amino acids

first step of translation cycle, tRNA is amino acylated

initiation

second step of translation cycle, mRNA and the aminoacylated tRNA bind to the small ribosomal subunit, the large binds as well

elongation

third step of translation cycle, successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until the ribosome reaches a stop codon

termination

fourth step of translation cycle, translation stops when a stop codon is encountered, the mRNA and protein dissociate and the ribosomal subunits are recycled

protein folding

fifth and final step of translation cycle

tRNA alignmnet

close locations of the ends of tRNAs in P and A sites allow for peptidyl transfer

translation initiation in bacteria

the 30s subunit binds IF-1, which blocks the A site, and IF-3, then the mRNA. the fMet-tRNA, accompanied by GTP-bound IF-2, base-pairs with the start codon. the 50S subunit associates caused by conformational change, the binding hydrolyzes GTP, and IF-1, IF-2, and IF-3 dissociate, leaving the complex.

IF-1

initiation factor that binds the ribosomal A site and blocks tRNA binding

IF-2

initiation factor that directs the initiating tRNA to the P site of the 30S subunit. the 50S subunit binds to the complex and hydrolyzes the GTP bound to IF-2, releasing it allowing the 70S subunit to form.

IF-3

initiation factor that prevents premature addition of the 50S subunit to the assembling initiation complex

initiation complex

complex of a ribosome with an mRNA and the initiating Met-tRNAi or fMet-tRNA, ready for the elongation steps

shine-dalgarno sequence

sequence in an mRNA that is required for binding bacterial ribosomes, also called ribosome binding site (RBS)

translation elongation in bacteria

the incoming aminoacyl-tRNA is bound by EF-Tu-GTP and inserted into the A site. GTP hydrolysis releases EF-Tu-GDP, leaving tRNA in place. tRNA base pairs with the mRNA codon and shifts into correct position so peptidy transfer can occur. EF-G facilitates translocation of tRNA

EF-Tu

elongation factor that delivers aminoacyl-tRNAs to the A site of the elongation complex with the help of GTP hydrolysis, it is a GTPase

EF-Ts

elongation factor that uses bound GTP to regenerate EF-TU-GTP from EF-Tu-GDP

EF-G

elongation factor with GTPase activity that facilitates the translocation of tRNAf rom the A site to the P and the P to E site during protein synthesis

kinetic proofreading of tRNA

tRNA ares kept if they are cognates, but rejected if they are wrong. codon/anticodon recognition is stable for correct matches and unstable for incorrect matches.

translocation

movement of ribosome by one codon along the mRNA

peptidyl transferase reaction

reaction that synthesizes the peptide bonds of proteins, nucleophilic attack of the alpha-amino group of the ribosomal A-site aminoacyl-tRNA on the carbonyl carbon of the ester bond linking the fMet to the P-site tRNA

translation termination in bacteria

when the ribosome comes to a stop codon, release factor 1 or 2 (1 for UAG, 2 for UGA and UAA) binds to the A site and induces release of the polypeptide chain. release factor 3 bound to GDP then binds to the ribosome and exchanges GTP for GDP, displacing the other release factor. GTP hydrolysis releases release factor 3.

class 1 release factors

codon specific, RF-1 - UAG, RF-2 - UGA and UAA

class 2 release factors

no codon specificity, GTPase, RF-3 binds to class 1 RFs and hydrolyzes GTP to promote their release

ribosome recycling

disassembly of translated mRNA, deacylated tRNAs a d ribosomal subunits in preparation for new rounds of translation

ribosome recycling factor (RRF)

bacterial factor involved in ribosome recycling that binds to the empty ribosomal A site and recruits EF-G to stimulate release of the deacylated tRNAs in the P and E site

kasugamycin

antibiotic that inhibits 30S subunit assembly and maturation, used in agriculture

puromycin

antibiotic that inhibits polypeptide synthesis by being incorporated into a growing peptide chain, causing its premature termination

tetracycline

antibiotic that inhibit protein synthesis by occupying the ribosomal A site, which prevents binding of aminoacyl-tRNAs

chromaphenicol

antibiotic that inhibits protein synthesis by bacterial, mitochondrial and chloroplast ribosomes by blocking peptidyl transfer

fusidic acids

antibiotic that stabilizes EF-G on the ribosomes after GTP hydrolysis

erthyromycin

antibiotic that binds and blocks the exit tunnel, not allowing protein chain to exit the ribosome

ricin

extremely toxic protein of the castor bean that inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific adenosine in 23S rRNA

ricin mechanism

chain a is catalytic, chain b is inhibitor. polypeptide chains linked by disulfide bond, upon incorporation bond cleaved. cleavage releases chain a which buried into ER membrane then translocated into cytosol. chain a hydrolyses N-glycosidic bond of the adenine residue resulting in a depurinated site. depruinated ribosome loses GTPase activation activity

alpha-sarcin

in a cell it cleaves 28S rRNA with high specificity, likely destabilizes membrane and spontaneously translocates through lipid bilayer

diphtheria toxin

bacterial toxin that catalyzes the ADP-ribosylation of a dipthamide residue of eEF2, thereby inactivating it and inhibiting protein synthesis by the eukaryotic ribosome, dimer of a and b chains

diphathamide

unusual modified histidine residue on the tip of eEF2, affected by diphtheria toxin

step 1 of eukaryotic translation initiation

ribosomal subunits are separated by eIF3 and eIF4, eIF1 binds to the E site

step 2 of eukaryotic translation initiation

GTP bound eIf2 binds to Met-tRNA and forms a ternary complex, interacts with eIF3, eIf1, eIFA to make 43S complex, eIF5 and eIF5B also associate

step 3 of eukaryotic translation initaition

complex eIF4F (made of eIF4E, eIf4A, eIF4G) regulates the binding of mRNA to the 43S complex

step 4 of eukaryotic translation initiation

the 43S complex scans the mRNA for the start codon, joining with the 60S ribosomal subunit to form the complete ribosome

step 5 of eukaryotic translation initiation

once the start codon is found, 80S ribosome is made, eIF5 stimulates hydrolysis of GTP on eIF2, eIF5B hydrolyzes GTP causing everything to disassociate

eIF3

prevents binding of premature ribosomal subunits

eIF1

binds to E site

eIF1A

prevents binding of tRNA to A site

eIF4E

binds to 5’ cap

eIF4A

is an ATPase and helicase

eIF4G

provides link by binding eIF4E and eIF3, binds PABP which brings 5’ and 3’ ends of mRNA together

IRESes (internal ribosomal entry sites)

site on the 5’ side of the start codon in some viral and eukaryotic mRNAs where a eukaryotic ribosome can bind in the absence of a 5’ cap

PRK

interferon-induced, double stranded RNA-activated protein kinase, part of innate immune response, induces global translational shut off and apoptosis

upstream open reading frames (uORFs)

short open reading frame upstream of a gene’s start codon that serves as a decoy to divert ribosomes, thereby down-regulating expression

viral strategies

prevent host mRNA synthesis, destabilize host mRNAs, prevent host mRNA translation through eIF4G cleavage and eIF4A inhibition

tmRNA

bacterial RNA that has the properties of a tRNA at its 5’ end and the properties of an mRNA. when aminoacylated the 5’ end can bind in the A site of a ribosome stalled ona truncated mRNA, and the 3’ end can serve as a template for continued translation through a termination codon that recruits the termination factors required for proper termination.

RNase-L

non-specific RNase that degrades everything possible to try and induce apoptosis, produced when virus is infecting and destroying

closed loop model

loop of mRNA that is caused by eIF4G bringing the 5’ and 3’ ends close together

TAP-tag

run cell extract over first IgG affinity column, which binds protein a tag. proteins that do not interact with target elute, cleave protein a with TEV proteases

GST-tag

insert gene for target protein and gene for gliutathione-S-transferase, run cell extract through column to remove other proteins, then add solution of free glutathione to column

sanger sequencing

if incorporated radioactive nucleotide (like ddATP) will get different length fragments from each place there is a Adenine.

pyrosequencing

thousands of beads each carrying different DNA are produced if correct nucleotide added it is incorporated and PPi is released which causes a flash of light