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Computational Biology I
LSM5191
Aylwin Ng, D.Phil
Lecture Notes:
Transcriptome:
Molecular Biology of Gene Expression II
TRANSLATION
RIBOSOMES: protein synthesizing machines
• Translation takes place on defined cytoplasmic organelles called
RIBOSOMES.
ROLES OF RNA IN TRANSLATION
Three types of RNA molecules perform different but complementary roles in
translation:
ƒ
Messenger RNA (mRNA) carries information copied from DNA.
ƒ
Transfer RNA (tRNA) deciphers the code and delivers the appropriate
amino acid specified by a codon (a unit of 3 nucleotides).
ƒ
Ribosomal RNA (rRNA) associates with a set of proteins to form
ribosomes, structures that function as protein-synthesizing machines
What is a Codon?
• A unit of 3 nucleotides.
• Each Codon (except for stop codons) encodes an amino acid.
TRANSFER RNA (tRNA)
• Forms a vital link between mRNA & the growing polypeptide chain.
• Anti-codon (on tRNA) base-pairs with CODON (on mRNA ).
• Amino-acid (a.a.) is attached at 3’-end of tRNA.
• Specificity: Each tRNA type is specific for only the a.a. it carries:
E.g.: tRNAMet carries only methione; and tRNAGly only glycine.
• 50 different tRNAs in eukaryotes.
• But only 20 amino acids are designated by the genetic code. Codon degeneracy.
Different tRNA types can carry the same amino-acid (due to ‘wobble’ basepairing).
• e.g. tRNAGly1 and tRNAGly2 are both specific for glycine.
• All mature tRNA ends with –CCA. (CCA added by tRNA nucleotidyl-transferase).
TRANSFER RNA (tRNA)
Cloverleaf structure
(dihydrouridine)
(ThymidinepseudoU-cytidine)
3-D Structure
Aminoacylation (‘charging’) of tRNA
• Attachment of amino-acid (a.a) to tRNA ensures specificity.
• Catalysed by aminoacyl-tRNA synthetases.
• Each tRNA is recognised by a specific aminoacyl-tRNA synthetase.
• Aminoacylation occurs in 2 steps.
Step 1:
• Formation of activated a.a.
intermediate;
• a.a linked to enzyme via highenergy bond.
Step 2:
a.a transferred to 3’-end of tRNA.
Overall rxn:
enz
a.a + ATP + tRNA Æ
Aminoacyl-tRNA + AMP + 2Pi
Step 1 Æ
Step 2 Æ
Codon & tRNA anticodon recognition
• Specificity of aminoacylation Æ ensures tRNA carries the right a.a. denoted by
the codon the tRNA pairs with.
‘Wobble’ base-pairing occurs
‘Wobble’ = non-standard (non-Watson-Crick) base pairing:
The ‘Wobble’ rules:
• G-U pairing acceptable.
• Inosine (I), [a modified version of Guanosine], can pair with A, C and U.
‘Wobble’ base-pairing
G-U base-pairing
Alanine codons
5’- GCC
GCU
GCA
GCG
Enables the 4 codons for alanine to be decoded by just
2 tRNAs.
Inosine base-pairs
with A, C and U
Enables the 3 codons for isoleucine to be decoded by
just one tRNA.
THE GENETIC CODE
TRANSLATION INITIATION
In Prokaryotes including bacteria,
ƒ Translation is initiated when the small ribosome subunit + initiation
factor (IF3) binds to Shine-Dalgarno seq. (5’-AGGAGGU-3’)
ƒ This seq. is 3-10 nucleotides upstream of the initiation codon (start
site).
ƒ Initiator tRNA is ‘charged’ with N-formylmethionine or methionine.
In Eukaryotes,
ƒ Ribosome binds to the 5’ end of mRNA by recognizing the
methylated cap.
ƒ Ribosome moves along mRNA until it encounters AUG within Kozak
seq (5’-ACCAUGG-3’) Æ initiation of translation.
ƒ Initiator tRNA is ‘charged’ with methionine.
TRANSLATION INITIATION (Eukaryotes)
Cap
5’
(A)n 3’
AUG
(A)n 3’
AUG
eIF4E
eIF4A
eIF4F
complex
tRNA
eIF4G
eIF4E
eIF2
40S
(A)n 3’
AUG
tRNA
eIF4F
complex
eIF4A
eIF2
40S
eIF4G
eIF4E
(A)n 3’
AUG
eIF2
60S
AUG
40S
(A)n 3’
Translation
ELONGATION OF TRANSLATION
• Mechanism very similar in bacteria and eukaryotes.
Peptide bond formation catalysed
by peptidyl transferase
eEF-1
Animation clip
EXERCISES
Exercise 1a:
5’- GTAGCCTACCCATAGG -3’
If mRNA is transcribed from this DNA using the complementary
strand as a template, what will be the seq. of the mRNA?
5’ – GUAGCCUACCCAUAGG - 3’
What peptide will be made if translation started exactly at the 5’ end of
this mRNA? (assume no start codon is req.d).
Valine(V) – Alanine(A) – Tyrosine(Y) – Proline(P)
Exercise 1b:
5’ – GUAGCCUACCCAUAGG - 3’
Potentially, how many different peptides are encoded in this mRNA?
3 different peptides, since there are 3 different reading frames.
5’ – GUAGCCUACCCAUAGG - 3’
V A Y P *
(Frame 1)
* P T H R
(Frame 2)
S L P I
(Frame 3)
Six peptides … if the stretch of DNA (in Exercise 1a) is also
transcribed in the opposite direction, i.e. both strands serving as
templates for transcription.
Exercise 2:
If the anti-codon of a tRNA has this sequence:
5’- G C U –3’
Which could be the likely corresponding codon sequence on the mRNA?
(1) 5’- C G A –3’
(2) 5’- A G C –3’
(3) 5’- C G T –3’
(2) and (4)
(4) 5’- A G U –3’
Which amino acid is the tRNA likely to be specific for?
Serine
Locating genes by scanning Open Reading
Frame (ORF)
Human Interleukin-2 (IL-2) gene
- promoter, exon 1 and partial cds (Accession Number: AF031845)
1
61
121
181
241
301
361
421
481
tatgacaaag
aaaactgttt
ctaatgtaac
aaattccaaa
gtctttgaaa
attaacagta
cacagtaacc
aagtcttgca
acaactggag
aaaattttct
catacagaag
aaagagggat
gagtcatcag
atatgtgtaa
taaattgcat
tcaactcctg
cttgtcacaa
catttactgc
gagttacttt
gcgttaattg
ttcacctaca
aagaggaaaa
tatgtaaaac
ctcttgttca
ccacaatgta
acagtgcacc
tgtatcccca
catgaattag
tccattcagt
atgaaggtaa
attttgacac
agagttccct
caggatgcaa
tacttcaagt
cccccttaaa
agctatcacc
cagtctttgg
tgttttttca
ccccataata
atcactcttt
ctcctgtctt
tctacaaaga
gaaaggagga
taagtgtggg
gggtttaaag
gactggtaaa
tttttccaga
aatcactact
gcattgcact
aaacacagct
What is the sequence of amino acids encoded by this piece of DNA?
But first, we need to know where the translational start site is.
There are eight possible initiation codons – which is the one?
Important: also need to find out the 5’-end of the mRNA transcript.
Human Interleukin-2 (IL-2) gene, exon 1
In Eukaryotes, scanning Open Reading
Frame (ORF) is complicated by Introns
Effect of Point mutations
Effect of Deletion mutations