Translation (Topic 2)

Amino acid features

20 different, central alpha carbon, amino group, carboxyl group, R-group, monomer of proteins

R group

distinctive side chain of amino acids, gives chemistry

peptide bonds

covalent bonds, join amino acid together, form polypeptide, between amino group and carboxyl group

4 levels of protein organization

primary, secondary, tertiary structure, quaternary structure

primary structure of protein

sequence of amino acids

secondary structure of protein

local regions of folding, hydrogen bonds/ Van der waals interactions/ hydrophobic effects, Alpha helices/ Beta-pleated sheets

Tertiary structure

3D shape of protein, stabilized by disulfide bonds (cysteine) and noncovalent interactions

Quaternary structure

multi-peptide protein complex, each polypeptide is subunit

Beadle and tatum

proposed one-gene-one-polypeptide, Neurispora crassa to determine genes controlled protein production

Genetic code

nucleotide sequence dicate amino acid sequence, degenerate/redundant, non ambiguous (one AA per codon), non overlapping, universal (used by most organisms)

codons

3 nucleotide (triplet) sequence, code for specific amino acid, 5’→3’

synonymous codons

code for same amino acid

Marshall Nirenberg and Heinrich Matthaei

deduce which codon responsible which amino acid, used synthetically produced RNA inserted in E. coli, won 1968 Nobel prize

Stop/termination/nonsense codons

3 codons (UAG/UGA/UAA), termination of translation

transfer RNA (tRNA)

brings amino acid to codons, ~75 nucleotides in length, contains anticodon (complemenatry to codon), 5’ CCA bind amino acid, allows wobble on 3 nucleotide in codon, 

Aminoacyl-tRNA synthetases

add amino acid to tRNA, 2 steps, 20 types (one each amino acid)

Aminoacyl-tRNA synthetases

carboxyl of AA reacts with alpha phosphate of ATP to form aminoacyl-AMP (release pyrophosphate), amino acid is charged to adenosine (of CCA) releasing AMP

ribosomes function

bind mRNA and identify start codon, faciliate base pairing between mRNA and tRNA, catalyse peptide bonds

bacteria ribosome

70S, 50S large subunit (23S + 5S), 30S small subunit (16S)

eukaryotes ribosome

80S, 60S large subunit (28S + 5.8S + 5S), 40S small subunit (18S)

3 sites of ribosome

aminoacyl-tRNA binding (A) site, peptidyl (P) site, exit (E) site

decoding center

ensures only tRNA with proper anticodon enter A site

peptidyl-transferase center

forms peptide bonds

aminoacyl-tRNA binding (A) site

binds incoming charged tRNA

peptidyl (P) site

forms peptide bond between amino acid, disconnects amino acid from tRNA in P site

exit (E) site

contains deacylated (uncharged) tRNA to be released

Translation initation

place fist charged tRNA into P site, always Met specified by AUG codon

Shine-Dalgarno sequence (ribosome binding site)

in bacteria, position small subunit to correct spot

Kozak sequences

in eukaryotes, surronds authentic start codon

translation initation in Bacteria

30S subunit binds IF3 (E site) and IF1 (A site), RBS aligns 30S on mRNA (30S initation complext), IF2 brings tRNAfMet to P site (use GTP), 50S subunit binds forming ribsomes

Translation initation in Eukaryotes

40S subunit bind eIF3(block large)/ eIF1A(blocks A site)/ eIF2 and 5B (charged tRNA to P), eIF4A/E/G forms complex (eIFAF), eIF4A binds 5’ cap/unwinds 5’ UTR/ use ATP scan for Kozak sequence - binds polyA binding proteins (PABP) (circularize mRNA), eIF dissociates allowing large subunit bind (eIF4F stays for next round of translations)

Translation elongation

Elongation factor (EF-Tu/EF-G or eEF1alpha/eEF2), charged tRNA associates with EF-TU and GTP (form ternary complex), enter A site causing ribosome shape change(displace P site), hydrolyze GTP and peptide bond forms, EF-G-GTP fits A site, hydrolyzes GTP causing shifting of tRNA down, EF-G leaves opening A site up, repeat

Translation termination

release factors (RF1/2 or eRF1/eRF3) mimic tRNA shape binding stop codon, water breaks peptide bonds (release polypeptide), RF3 release RF1/2, GTP hydroyzles cause RF3 and ribosome dissociates

Ribosome recycling factor (RRF)

binds A site with IF3, prepares for next round

Post-translation modification

folding guided by chaperone proteins, AA side chain modoification (phosphates, disulfide, cleave smaller, tag for export)