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Lecture 12-30 Transcription Translation
Transcription
1
Transcription Translation
Translation
Protein Synthesis Part 1: Take Home Message
Required reading: Stryer Ch. 34, p. 875-888 Ch. 33 p. 849-850
1) Translation of the genetic code is dependent on three base words that correspond to a single
amino acid AA (codons).
2) The mRNA message is read by tRNA through the use of a three base complement to the three 3
base word (anticodon).
3) A specific amino acid AA is conjugated to a specific tRNA.
4) Amino acid AA side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases
to conjugate a specific three 3 base message with a specific amino acid AA.
Lecture 12-30 Transcription Translation
2
Table 1. The genetic code. for mRNA 
1st position
(5' end)
U
C
A
G
Genetic Code
2nd position
3rd position
(3' end)-->
U
C
A
G
Phe
Phe
Leu
Leu
Ser
Ser
Ser
Ser
Tyr
Tyr
STOP
STOP
Cys
Cys
STOP SelenoCys Trp Mitochondria
Trp
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
His
His
Gln
Gln
Arg
Arg
Arg
Arg
Ile
Ile
Ile
Met init
Thr
Thr
Thr
Thr
Asn
Asn
Lys
Lys
Ser
Ser
Arg
Arg
Val
Val
Val
Val
Ala
Ala
Ala
Ala
Asp
Asp
Glu
Glu
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of
protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into
valine and glutamic acid, respectively. Note that those codons with
specify the more hydrophobic amino acids AA.
U or C as the second 2 nucleotide tend to
Lecture 12-30 Transcription Translation
3
Structure of Transfer RNA
Amino Aci d O H
Conjugati on A 3'
Site C
5' P
C
O H +H
C N
H
O C
Du pl ex
A 3' R
C
regi on
C
DHU l oop
T  C l oop
C
A
G
T  C
G
G
O
Du pl ex regi on
H
H2C
H2C
N
N
O
O
HN
di-hydro-uri dine (DHU)
NH
N
O
ps eu do-uri dine ( )
U
Di-hydro-uridine (DHU)
Anti Codon
Pseudo-uridine ()
Classes of Aminoacyl-tRNA Synthetases
• Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger Amino Acids)
• Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr (Generally the smaller amino acids)
H
H
tRNA
N
1
N 5
O
6 N
7
4
2
O P O 5'
8N
N
H O
3
9
O H
4' H
Charged tRNA -76 -76
1'
H
3'
H
O
C
O C
H
2' H
H
tRNA Activation by aminoacyl tRNA synthases
1. Amino-acyl-AMP formation
+
N H
H
Lecture 12-30 Transcription Translation
H O
H O
O
O
R
P
P
O
O
O
O
H
O
O
+ C
O
O
P
P
Adenine
+
O H
N
C
O
R O
H
P
O
O
H
H
O
+ O H H
Adenine
O
+ C
O
O
O
P
H
N
H H
C
O
H
H O
O
O
H
H
H
O
O
O
H
H
O
+ PO
OH
H O
H
H
O
O P
H
H
OH
O
H O
4
2. Aminoacyl transfer to the appropriate tRNA + AMP1-
R
H
H
H O
+
O
N CC
H
O
O
H
O
Adenine
O
P
O
H
H
H
O
H
+
O ACC
OH
Adenine
O
P
t RNA
R
H
H
O
+
N CC
H
H
O
O
O
+
O ACC t RNA H
H
H O
H
H
OH
Lecture 12-30 Transcription Translation
5
tRNA Recognition by Synthetases
• Specific recognition of the anticodon; ex. an change the anticodon for tRNATrp to
that for methionine and get good acylation by tRNAMet.
•Stem sequences can be crucial; ex. tRNAAla depends on GC at position 3:70 doesn't care what the codon is.
• Both the stem regions and anticodon are needed; ex. tRNA Gln.
tRNA Recognition by Synthetases
tRNA-Ala
tRNA-Phe
Trna- Ser
OH
A 3'
C
C
OH
A 3'
5' P
C
C
5' P
OH
A
C
C
5' P
3'
U70
G3
C
T C
G
A
C
T C
G
A
GG
GG
A
C11
C
T C
G
GG
G24
U
U
G34
A35
A36
U
tRNA Recognition by Synthetases
 S tem and ATP S ite
t-RNA 
Anticodon Recognition 
 Additional Recognition
Lecture 12-30 Transcription Translation
6
tRNA Synthetase Proofreading Ile
Large
Smaller
Acylation Site Hydrolytic Site
Large
Smaller
Acylation Site Hydrolytic Site
H
H
H
C
H H
HC
H H
C
H
H
+
C
O
O
H N
H
H
C HH
C
N
C
C
Ile
t RNA
O

t RNA
Difference 
Ile
+H
H
H
O
H
H
C H H
H
C
H
H
+
H N
C
H
O
C
C
O
C
H
H H
H H
C
C
O
t RNA
Correct Acylation
Isol eucine
Isol eucine
H
H
+
N H
t RNA O
H
Mis-acylation
tRNA Synthetase Proofreading Val
Hydrophobic
Polar
Acylation Site Hydrolytic Site
H
H
H C
H
H
H
N
C H
H
H
H
O
+
O
H
H
+
N H
V al
t RNA
O
H
H
H
C
O
H

Difference 
t RNA
H H
CH
C
H
V al
O
H
H
C
H
O H
H
H
H
Hydrophobic
Polar
Acylation Site Hydrolytic Site
O
+
O
N
H
O
Correct Acylation
H
+
N H
H
t RNA Valine
Valine
t RNA O
Mis-acylation
Lecture 12-30 Transcription Translation
7
How do we go from mRNA to Protein?
Incoming tRNA
Table 1. The genetic code. for mRNA 
1st position
(5' end)
U
C
A
G
Genetic Code
2nd position
3rd position
(3' end)-->
U
C
A
G
Phe
Phe
Leu
Leu
Ser
Ser
Ser
Ser
Tyr
Tyr
STOP
STOP
Cys
Cys
STOP SelenoCys Trp Mitochondria
Trp
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
His
His
Gln
Gln
Arg
Arg
Arg
Arg
Ile
Ile
Ile
Met init
Thr
Thr
Thr
Thr
Asn
Asn
Lys
Lys
Ser
Ser
Arg
Arg
Val
Val
Val
Val
Ala
Ala
Ala
Ala
Asp
Asp
Glu
Glu
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of
protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into
valine and glutamic acid, respectively. Note that those codons with
specify the more hydrophobic amino acids AA.
Codon : Anticodon
First 1st Base of Anticodon is Variable
3 2 1
t RNA- 3'-X Y Z- -5' anticodon
mRNA- 5'-XΥYΥ ZΥ -3' codon
1 2 3
Third 3rd Base of Codon is Variable
U or C as the second 2 nucleotide tend to
Example tRNAPhe
3Υ--A--A--GMe -5Υ
5Υ-U - U - C -3Υ
3Υ--A--A--GMe -5Υ
5Υ-U - U - U -3Υ
Lecture 12-30 Transcription Translation
8
Wobble pairing rules
5’ anticodon base
3’ codon base
C
G
A
U
U
A or G
G
C or
U
U , C, or A
I
tRNA Anticodon-Codon Recognition
Anticodon 3’ – C – G – I – 5’
3’ – C – G – I – 5’
3’ – C – G – I – 5’
U – 3’
5’ – C – G – C – 3’
5’ – C – G – A – 3’
Anticodon 3’ – C – G – G – 5’
3’ – C – G – G – 5’
U – 3’
5’ – C – G – C – 3’
Anticodon 3’ – C – G – U – 5’
Codon
5’ – C – G – A – 3’
3’ – C – G – U – 5’
5’ – C – G – G – 3’
Anticodon 3’ – C – G – C – 5’
3’ – C – G – A – 5’
Codon
5’ – C – G –
Codon
5’ – C – G –
Codon
5’ – C – G –
5’ – C – G – G – 3’
U – 3’
tRNA Anticodon-Codon Recognition
Adenosine
H
H
N
N
N
Inosine
Guanosine
O
O
H
N
N
N
N
H
N
N
N
Ribose
N
H
N
N
N
H
H
Anticodon 3’ – C – G – I – 5’
3’ – C – G – I – 5’
3’ – C – G – I – 5’
Codon
5’ – C – G – A – 3’
5’ – C – G –
N
N
¥C 1 N
5’ – C – G – C – 3’
O HN H
NH N
N
O
¥C 1
¥C1 N
I------C base pair
N
N
O HN
N
N
NH N
N
U – 3’
O
H
H
N
O
N
H
N
H
¥C1
I-----A base pair
NH O
N
¥C1
¥C1 N
I----- U base pair
Nonsense suppression
Nonsense mutations = change of codon for an AA to S TOP
Usually lethal Πtruncated protein
Can be rescued by mutation in a different part of the genome
Mechanism: tRNA gene mutation
Example: E. Coli Amber suppressor
tRNATyr anticodon change G U A C U A
Mutated tRNA recognized S top codon as
Tyr and prevents chain termination
Lecture 12-30 Transcription Translation
9
Table 1. The genetic code. for mRNA 
1st position
(5' end)
U
C
A
G
Genetic Code
2nd position
3rd position
(3' end)-->
U
C
A
G
Phe
Phe
Leu
Leu
Ser
Ser
Ser
Ser
Tyr
Tyr
STOP
STOP
Cys
Cys
STOP SelenoCys Trp Mitochondria
Trp
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
His
His
Gln
Gln
Arg
Arg
Arg
Arg
Ile
Ile
Ile
Met init
Thr
Thr
Thr
Thr
Asn
Asn
Lys
Lys
Ser
Ser
Arg
Arg
Val
Val
Val
Val
Ala
Ala
Ala
Ala
Asp
Asp
Glu
Glu
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of
protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into
valine and glutamic acid, respectively. Note that those codons with
specify the more hydrophobic amino acids AA.
U or C as the second 2 nucleotide tend to
Protein Synthesis Part 2: Take Home Message
1) Translation always proceeds by reading the mRNA sequence from the 5’ to the 3’ end.
2) Amino acids are assembled in to proteins with the first amino acid having a free amino group
—N
3
+
(N-terminus) and the last having a free carboxylate—COO— (C-terminus).
3) Synthesis of the protein is processive and contains steps;
a) initiation, b) elongation and c) termination
Lecture 12-30 Transcription Translation
Overall Ribosome Structure:
10
Translation Machine
23S RNA

50S S ubunit 
34 proteins 
5S RNA
16 S RNA
21 proteins
30S S ubunit 
30S S ubunit 
70S Ribosome
Large subunit 50 S ¥ 70S can be dissociated into two 2
subunits: 50S and 30 S
¥ S mall subunit (30S ) contains 16S RNA
and 21 proteins
¥ Large subunit (50S ) contains 23S RNA,
5S RNA, and 34 proteins
¥ 50S and 30S can come back together
to form 70S
¥ rRNA is folded and has distinct
tertiary 3΅ structure
¥ rRNAs probably dominate the ribosome
function, 23S necessary for
catalytic function
S mall subunit 30 S
• tRNA site, peptidyl transferase, GTPase and Exit site

 50S S ubunit


Lecture 12-30 Transcription Translation
11
T T --GA T -- T C T -AAA-- T AA- 3’ Translating the Message
DNA
5'-ATG---GCC- T
RNA
5' -A U G--GCC- U U U --GA U -- U C U -AAA-- U AA--3’
Protein
N- Met
5'
Elongation
Ala
Phe
Asp
Ser
Overview of Translation
Lys Stop -C
DNA
RNA Pol
{
Stop
Protein
50S
Ri bosomes
3'
30S
¥ M ultiple ribosomes can be
involved simultaneously, max.
density one 1 ribosome per
80 nucleotides
¥ All mRNAs contain signals for
starting and ending
polypeptide synthesis.
70S
mRNA
5'
Initiation
Protein Synthesis
Part 1:
Take Home Message
1) Translation of the genetic code is dependent on three base words
that correspond to a single amino acid.
2) The mRNA message is read by tRNA through the use of a three base complement to
the three 3 base word.
3) A specific amino acid is conjugated to a specific tRNA (three base word).
4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to
conjugate a specific three base message with a specific amino acid AA.