LECTURE 3

Learning Objective:
 Describe the general steps of mRNA processing in eukaryotes.
 Draw and label a ribosome showing the key features for translation.
 Explain translation in a Eukaryote cell, including initiation, elongation and termination.
 Discuss the key steps that ensure that the correct amino acid is included in the peptide chain.

RNA
 RNA synthesis
o Basic chemistry is same as DNA synthesis
o Catalysed by RNA polymerase
 Base pairing differs
 T=A
 G=C or C=G
 A=U
o A=U base pairing is the same as A=T
o Little difference between thymine and uracil group
o
One less methyl
group

Transcription Topology
 Gene sits within DNA – double strand
 Differentiate between template and non-template (coding) strand
 Usually oriented from 5’ to 3’ direction
o Transcription uses opposite strand as template
o Due to RNA transcription happening from 5’ to 3’
 Transcription start upstream than coding sequence
o Everything upstream is where promoter etc. are
 Start at +ive going downstream and -ive going upstream
 Transcript we start with is primary and need to go through steps to be ready (capping etc.)
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Eukaryotic mRNA Processing

 The 5’ cap
o Protects mRNA from degradation
o Involved in nuclear export/translation
o 5’-PPP is an immune trigger
 Added shortly after initiation
o After 25b have been produced
o Linked to elongation
o (ieNo Cap –No mRNA)
 Cleavage
o Site at end of DNA signals RNA to stop and cleave off
 Poly(A) tail to use protein
 RNA splice extrons

Translation
 Translation: convert mRNA info. into protein by polymerisation of amino acids
 Core structure of amino acid with R group
o R group alters size, charge etc. giving different functions
o Arginine positive can bind to DNA and RNA which have negative charge
o Tryptophan
 Condensation reaction forms protein and peptide bond
o Ribosome accelerates reaction
 mRNA codes for 20 amino acids
o codons code for specific amino acids
o code is degenerate – not every codon codes for every amino acid
o position ribosome in right place – frame is important
 tRNA
o bridge between mRNA and protein
o tRNAs use Watson-Crick base pairing at position 1 and 2
o acceptor stem: where amino acid is conjugated to
o codon (in mRNA) and anti-codon (in tRNA) pairing determines the amino acid that forms in the
next part of chain
 anticodon – complementary base pairing with codon that gives correct reading of mRNA
 codon read in 5’ to 3’ direction and pairs in antiparallel manner
 5’ nucleotide of anti-codon binds to the 3’ position of codon
o Position 3 in codon is called the wobble position - degenerate
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 Use non-standard pairing : G-U, C-I
o One tRNA to several codons – codon code is degenerate
 E.g . four codons to one anticodon
o tRNA are covalently linked to appropriate amino acid by an aminoacyl-tRNA synthase specific
for a particular amino acid
 proof-reading for translation so correct amino acid is paired with correct tRNA
 each enzyme specific for single amino acid – recognise the stem region of tRNA with
specific amino-acid paired with particular anticodon

Translation: 3 Steps
 Ribozyme: 2/3 rRNA and 1/3 protein

 Initiation (loading a ribosome onto mature mRNA in the correct place and finding the start site)
o Eukaryote Initiation factors (eIF’s) interact with mRNA and Ribosome
o eIF4 complex recognises the 5’Cap of mRNA and poly(A) binding protein
 have circular version of transcript
 multiple components:
1. eIF4A: RNA helicase to remove any secondary structure from the mRNA
2. eIF4B: has 2 RNA binding sites, one nonspecific site binds the mRNA the second is
specific from the 18S rRNA component of the ribosome
o first component of ribosome to be loaded onto mRNA is 40S subunit
 loaded as 43S preinitiation complex: 40S subunit with eIF3, eIF1, eIF1A and eIF5
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 MET-tRNA included with eIF2 that has GTP bound to it
 43S preinitiation complex attaches to the mRNA and scans from 5’ to 3’ to identify
starting codon
o Binding between start codon and MET-tRNA triggers GTP hydrolysis (to GDP) by eIF2 (energy
source)
 Identifying the correct start site
 Kozak sequence 5’-GCC-Pu-CCAUGG-3’ – always in genome
o Recognised by small subunit
 Required for efficient translation
o Allow 60S subunit to join and displace initiation factors with eIF5B (with bound GTP) and eIF1A
remaining in the aminoacyl tRNA binding site
o Correct pairing of large and small subunit trigger hydrolysis of GTP by eIF5B and eIF1A released
o 80S initiation complex ready to undertake elongation
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 Elongation (read mRNA – associate codon with an amino acid – repeat)

o 3 tRNA binding sites
 Aminoacyl – incoming tRNA
 Peptidyl – peptide bond formation occurs
 Exit – tRNA exit
o Incoming aminoacyl-tRNA delivered as complex with EF1α (tRNA always with cofactor)
 EF1α bind to the tRNA acceptor stem and amino acid and GTP
 Not specific for particular amino acid
o tRNA bound in aminoacyl tRNA site with correct codon-anticodon pairing at decoding site
 ribosome triggers hydrolysis of GTP bound to EF1α - released
 ribosome samples the interaction to ensure correct tRNA is used
o The tRNA relaxes into the A-site with the amino acid located in the Peptidyl Transfer Centre
(PTC) in a process called accommodation
o New amino acid positioned in PTC, peptide bond form
 Ribosome gives optimal environment for reaction to occur – doesn’t participate in
reaction
o Following Peptide bond formation tRNA’s occupy hybrid sites with a single tRNA bound in
the decoding site and PTC.
 tRNA located in decoding site and old tRNA leaning into exit site
o Elongation factor 2 (EF2) uses GTP to push tRNA’s through ribosome
 mRNA is dragged along by the tRNA
o tRNA’s now occupy the E and P sites.
o A-site is free to accept new aminoacyl-tRNA for next codon.


Termination
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o Eukaryote Relase Factor 1 (eRF1) recognises all stop codons and use GTP to cleave peptide
site
o eRF3 associates and hydrolyses GTP to release polypeptide chain
o Components of Ribosome are recycled