Prokaryotic and Eukaryotic protein synthesis

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4 stages of protein biosynthesis?

-activation of individual amino acids
-initiation of synthesis
-elongation of the peptide chain
-termination of synthesis
all of the steps except the first one, occurs on the ribosome

activation of amino acids

-amino acid+ATP-->aminoacyl-AMP
-aminoacyl-AMP+tRNA-->aminoacyl-tRNA
no additional energy needed to make peptide bond but GTP is required for the ribosome to function

Fidelity of protein synthesis (aninoacyl-tRNA synthetases)

-accuracy of protein synthesis is dependent on aminoacyl-tRNA synthetase, whatever is linked to the tRNA is inserted to the protein
-proofreading of tRNA charging: amino acyl tRNA synthetases have a second active site that proof reads and hydrolyzes incorrectly linked amino acids from tRNA
-error rate of protein synthesis is not nearly as low as that for DNA replication
-proteolysis destroys any protein mistakes so they arent passed onto progeny

Ribosome

-half protein half RNA
-Eukaryotic is 80s, prokaryotic is 70s
-can dissociate into two ribonucleoprotein subunits (large and small)
-Eukaryotes (80s) have a 40s and 60s subunits
-Prokaryotes (70s) have a 50s and a 30s subunit

tRNA binding sites of the ribosome

Exit site: holds leaving amino acid free tRNA
Peptidyl site: binds Met-tRNA at initiation and peptide linked tRNA during elongation
Acyl site: new aminoacyl tRNA is added during elongation

Prokaryotic protein synthesis: Initiation

-the mRNA is aligned over P site when the 16s rRNA of the 30s (IF3 prevents premature assembly of the 30s and 50s subunits) subunit recognizes the Shine-Dalgarno sequence near 3' end
-IF2 binds GTP and N-formyl Met-tRNA, and IF1 is a stimulatory factor
-IF1 and IF2 are released when the 50s subunit combines and the GTP is hydrolyzed
-initiation occurs at start sites far from the end of the mRNA

Prokaryotic protein synthesis: Elongation

-new tRNA binds to the A site (involves complex containing charged tRNA, EF-Tu, and GTP.....upon binding the GTP is hydroyzed and EF-Tu*GDP is released from ribosome)
-formation of a peptide bond by peptidyl transferase (rRNA may be a catalyst), the alpha amino group at the A site acts as the nucleophile, and the tRNA at the P site acts as the leaving group
-translocation of the ribosome to the next codon, tRNA is shifted form the A site to the P site, requires GTP

Energy input for Elongation

-2 GTPs are used by the ribosome
-energy derived from the activated aminoacyl-tRNA is used to make the peptide bond

EF-Ts

-needed to convert EF-TuGDP to EF-TuGTP

elongation factors in prokaryotes (eukaryotic homologues are in parentheses)

-EF-Tu (EF1): loads next aminoacyl-tRNA into A site
-EF-Ts: (EF1beta)recharges loading factor (EF-Tu) with GTP
-EF-G: (EF2)advances ribosome to next codon

Prokaryotic protein synthesis: Termination

-termination codons signal termination (UAA, UAG, UGA)
-RF1 recognizes UAG, UAA & RF2 recognizes UGA, UAA
-RF3 is a G protein related to EF-Tu, and it mediates interactions between RF1 and RF2
-Eukaryotes have only one release factor, eRF, and it is also GTP dependent
-the appropriate release factor binds directly to termination codon and hydrolyze the protein-tRNA ester linkage by providing a water molecule to the peptidyl transferase site

Polysomes

-look like beads on a string
-multiple ribosomes attached to mRNA

a major difference between protein synthesis between bacteria and eukaryotes

transcription (DNA to mRNA) and translation can happen at the same time in bacteria but not in eukaryotes

principle behind antibiotics

differences between prokaryotic and eukaryotic ribosomes allow some compounds to block protein synthesis in bacteria and not in humans

streptomycin

cause misreading of mRNA

tetracyclin

binds to 30s and inhibits binding of aminoacyl-tRNA, could affect eukaryotic ribosomes but most effective against prokaryotes

chloramphenicol

inhibits peptidyl transferase activity of 50s, will also block mitochondrial protein synthesis

cycloheximide

inhibits peptidyl transferase activity of 60s

erythromycin

binds to 50s and inhibits translocation

puromycin

causes premature chain termination

new antibiotic approach

-synthetic compounds that bind at the site of interaction between small and large ribosomal subunits

Linezolid (Zyvox)

bind to the 23s RNA of large subunit and blocks 30s subunit binding, has been useful in treating multiantibiotic resistant gram positive infections, some Enterococcus strains have developed resistance via 23s rRNA mutations

Eukaryotic protein synthesis: Initiation

-eIF6 aids the disassociation of 80s to 40s and 60s, and is involved in the synthesis of 60s
-three complexes are formed
-43s complex: 40s subunit forms a complex with eIF3 and eIF4C
-mRNA complex: eIF4-mRNA complex: involves eIF4G, eIF4E which binds the 5'cap, PABP which binds the poly A tail, and eIF4A which is an RNA helicase
-Ternary complex: eIF2 delivers Met-tRNA to the 40s as an eIF2, GTP, and Met-tRNAf complex

Eukaryotic initiation occurs when-->

the first AUG codon is encountered, as the ribosome moves in a 3' direction away from the 5' cap structure, scanning to the first AUG requires ATP

Regulation of eukaryotic initiation

-controlling eIF-2 availability by posphorylation at serine 52

Heme regulated inhibitor, HRI

-low levels of heme in reticulocytes activates a kinase that phosphorylates eIF2
-if HRI is deficient, it can lead to different types of anemias

Unfolded protein response

excess unfolded proteins in the ER lumen will activate PKR-like ER kinase (PERK) that will reduce rate of protein synthesis by phosphorylating eIF2

eIF2 regulation as an antiviral mechanism

-type I interferons are synthesized in response to double stranded RNA, and interaction of interferons with their receptors will lead to expression of PKR, which forms an active dimer that phosphorylates eIF2 on serine 51, resulting in attenuation of protein synthesis (viral and cellular)
-interferons also lead to activation of RNase L, which degrades mRNA-->inhibition of protein synthesis
-activation of PKR also leads to phosphorylation of IkB, which leads to gene transcription by NF-kB

Viral evasion responses to Interferon-PKR response

-inhibit PKR activation
-mask the presence of dsRNA
-dephosphorylate eIF2

viral inhibition of PKR activation

-adenovirus makes a partially dsRNA which binds PKR and blocks dimerization
-vaccinia makes a pseudo substrate for PRK, called K3L which binds to and inhibits the kinase (K3L has structural similarity to eIF2)

virus ability to mask the presence of dsRNA

-make dsRNA binding proteins that inhibit PKR activation
-done by influenza, vaccinia, herpes simplex
-some of these proteins can directly interact with PRK and prevent dimerization

viral desphorylation of eIF2

-herpes simplex protein can combine with a cellular phosphatase and remove phosphates from eIF2

Picornoviruses: RNA translation

-blocks cellular protein synthesis by removing amino terminal 1/3 of eIF4G, leads to loss of binding sites for cap and poly A tail recognition proteins, now viral message containing IRES aids 40s to exclusively translate viral mRNA

Rotavirus: RNA translation

-mRNAs are capped but not polyadenylated, virus makes the NSP3 protein that competes with PABP (poly A binding protein) for eIF4G binding, NSP3 interacts with a sequence at 3' end leading to preferential translation of viral messages

Adenovirus: RNA translation

makes a protein that is able to displace eIF4E kinase Mnk1, lack of eIF4E phosphorylation reduces the translation of capped cellular mRNAs and increases capped viral mRNAs

Encephalomyocarditis virus: RNA translation

viral protein causes the dephosphorylation of 4E-BP1 which then forms a complex with eIF4E, the sequestration of eIF4E reduces cellular translation and increases viral translation

Diptheria toxin

inhibits eukaryotic protein synthesis by catalyzing a dipthamide residue in EF-2 by modifying a histidine residue
NAD+ is used

Ricin and Abrin

plant ribosome inactivating enzymes
-RNA backbone is not broken but a single base is released and that inactivates translation, it resembles N-glycosidase activity

Puromycin

resembles 3' end of a charged tRNA, becomes linked to growing peptide chain then terminates its extension
-binds to A site, shifts to P site and releases incomplete peptide chain
-works on both prokaryotes and eukaryotes

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