Lecture Notes

Week 8 Notes: Translation

PART I: Recap
 DNA in nucleus contains genes that are transcribed to make mRNA. Pre-mRNA produced and processed:
introns spliced, ends are added and the resultant mRNA is exported to cytoplasm, ribosome translates
mRNA to produce polypeptide

 Proteins synthesis in cytoplasm requires 3 types of RNA:
o mRNA: carries genetic info
o tRNA: deciphers the codons
 conformation of tRNA results from base pairing (H bonding) within molecule
 contain non-standard bases due to modification after transcription
 3’ end is the amino acid attachment site: binds covalently. Always CCA
 Anticodon: site of complementary base pairing with mRNA. Unique for each species of
tRNA
 Amino acid is attached to 3’ end (CCA) of tRNA by an energy-rich ester bond. This
provides energy for synthesis of peptide bond to join amino acids.
o rRNA: associates with set of proteins to form ribosomes

 There are more codons than there are different amino acids in proteins
o Combinations of the 4 bases give 64 different codons for 20 amino acids

Introduction
 A ribosome binds mRNA and tRNA. Ribosomes are ribonucleoprotein particles comprising 2 subunits.
o Each subunit contains a major rRNA and a number of small proteins

Translation occurs by initiation, elongation and termination
AA brought to ribosome by aminoacyl-tRNA and added to nascent peptide chain by interaction with the tRNA that
brought the previous AA

 Ribosome has 3 tRNA binding sites:
o aminoacyl-tRNA enters A site.
o Peptidyl-tRNA (tRNA carrying the nascent pp chain) is bound in the P site
o Deacylated tRNA (lacking AA) exits via E site

 Translocation: AA added to pp chain by transferring pp from peptidly-tRNA in P site to aminoacyl-tRNA in
A site

 Intiation: small + large subunits + aa-tRNA (translation stages up to synthesis of first peptide bond)
 Elongation: ribosome moves along mRNA, pp chain extended by addition AA
 Termination: translation ends by stopping AA addition and causing disassembly of synthetic machinery

Special mechanisms control the accuracy of translation
 Error rates differ at each stage of gene expression
 Wrong base, wrong tRNA, frameshift, wrong AA, wrong aminoacyl-tRNA (increasing error rate)

Initiation in bacteria needs 30S subunits and accessory factors
 Ribosome binding site: bacterial mRNA sequence that includes initiation codon that is bound by 30S
subunit during initiation
 Initiation is not a function of intact ribosomes, it requires separate 50S and 30S subunits which
reassociate during initiation
 mRNA recognition occurs when 30S subunit carrying initiation factors binds to form initiation complex
at RBS.
o When initiation complex forms, IF-3 is released to allow 50S to join to form complete ribosome

 Initiation factors (IF-1, IF-2, IF-3) bind to 30S subunits
o IF-3
 stabilises free 30S subunits
 inhibits premature binding of 50S subunit

, Week 8 Notes: Translation

 enables 30S subunits to bind initiation sites
 checks accuracy of recognition of first aminoacyl-tRNA
o IF-2
 Binds special initiator tRNA and controls its entry into ribosome P site
o IF-1
 Binds in the vicinity of A site, preventing aminoacyl-tRNA from entering

Initiation involves base pairing b/w mRNA and rRNA
 Bacterial mRNA initiation site: AUG initiation codon preceded by Shine-Dalgarno polypurine hexamer
o Shine Dalgarno: polypurine sequence AGGAGG centered about 10 bp before AUG codon on mRNA
 30S bacterial ribosomal subunit rRNA (3’ end of 16S rRNA) has complementary
sequence that bp w/Shine-Dalgarno seq during initiation

Initiator tRNA (tRNAfMet) starts the pp chain
 Translation starts with Methionine AA coded by AUG
 Different Met tRNAs in initiation & elongation
o Only tRNAfMet (controlled by IF-2 & ribosome) can be used for initiation by 30S subunits
 IF-2 binds tRNAfMet and allows entry into partial P site on 30S subunit
o Other aminoacyl-tRNAs must be used for elongation by 70S ribosomes

 N-formyl-methionyl-tRNA (tRNAfMet): aminoacyl-tRNA that initiates bacterial pp translation
 Amino group of the Met is formylated
 initiator tRNA has unique structural features that distinguish it from other tRNAs
o NH2 of Met bound is formylated (tRNAfMet)
o Bases at the last position of the stem are unpaired
o 3 G-C bp required to allow tRNAfMet to be inserted directly into P site

 tRNAmMet: bacterial tRNA that inserts Met at internal AUG codons

Small subunits scan for initiation site on eukaryotic mRNA
 40S subunit binds mRNA 5’ end and scans mRNA until initiation site reached
o Initiation site: 10 nucleotide sequence (Kozak sequence) that includes AUG codon
 60S subunit joins the complex at initiation site
 IRES (internal ribosome entry site): eukaryotic mRNA sequence that allows ribosome to initiate pp
translation w/o migrating from 5’ end

Eukaryotes use a complex of many initiation factors
 IFs required for all stages if initiation including binding the initiator tRNA, 40S subunit attachment to
mRNA, movement along mRNA and joining of the 60S subunit
o At least 12 in eukaryotes
 Initiator tRNA is Met-tRNA, different from Met-tRNA used in elongation b/c Met is not formylated
 Some eIFs bind to 40S subunit to form 43S preinitiation complex
 Cap-binding complex binds to mRNA 5’ prior to mRNA association with 40S subunit
 43S complex binds mRNA and scans for initiation codon, forming the 48S complex

 eIF2 binds initiator Met-tRNA & GTP, forming a ternary complex that binds to 40S before associating
w/mRNA
o eIF3 maintains free 40S subunits
o eIF2 binds Met-tRNA to 40S. eIF2 is a GTPase, eIF2B is the exchange factor.

 eIF4F is a heterotrimer consisting of:
o eIF4G: a scaffold protein that binds two further factors
o eIF4E: binds 5’ methyl cap
o eIF4A: helicase that unwinds the 5’ structure
 stimulated by eIF4B

 PABP binds 3’ poly(A)