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Lecture 11-30 Transcription Translation
Transcription
1
Transcription Translation
Translation
Antisense therapy: a new generation of drugs?
Lecture 11-30 Transcription Translation
2
Potential advantages of antisense drugs:
1.Specificity.
2.Facilitated drug development process.
3.Reduced side effects.
Problems associated with antisense drugs:
1. Delivery through cell membranes.
2. Degradation with nucleases.
3. Possibility of severe side effects in some individuals.
Figure 2. Therapeutic Nucleic Acids
Possible chemical modifications of a therapeutic
nucleic acid are virtually limitless. In natural DNA
(top left). Each backbone consists of ribose sugars
bridged by phosphate groups. The backbone
supports genetic code, a succession of bases.
Here two 2 are seen: thymine, the coce letter T,
which makes two 2 hydrogen bonds (orange arrows)
with its complement, adenine A (not shown):and
guanine G, which makes three 3 hydrogen bonds
with its complement, cytosine C.
Replacement of oxygen O by sulfur S in each phosphate group (above) makes the beckbone resistant to
nucleases, which would otherwise cut the bridges. However, the macromolecule remains electrically charged,
impeding its passage across cell membrane. The entire backbone can be replaced with a chain resambling
protein (bottom). The result is nuclease-resistant and the bases retain correct alignment. However, this
construct, too, does not readily cross cell membrane.
Lecture 11-30 Transcription Translation
3
Vitravene (Isis Pharmaceuticals, 1998)
 used against cytomegalovirus (herpes infection on the eye) in AIDS patients
 Phospho-thiolate oligonucleotide: 5’-GCG T
T T GC T C T T C T T C T T GCG-3’
 Mechanism: binds mRNA coding for the protein required for viral replication and causes its degradation
with RNAase H.
Lecture 11-30 Transcription Translation
4
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 base word
(anticodon).
3) A specific amino acid is conjugated to a specific tRNA.
4) Amino acid AA side chain size, hydro-phobicity and polarity govern the ability of tRNA synthetases
to conjugate a specific three base message with a specific amino acid AA.
How do we go from mRNA to Protein?
DNA  mRNA  Proteine
t-RNA  Amino Acid Sequence
Translating the Message
DNA
RNA
Protein
5'-A T G-GCC- T
T T -GA T - T C T -AAA- T AA-3'
5'-A U G-GCC- U U U -GA U - U C U -AAA- U AA-3'
N-- met
ala
phe
asp
ser
lys
stop -C
Translation of mRNA
Sequence of 3 Bases = One Codon
One Codon = One Amino Acid AA
20 Natural Amino Acids AA = 64 Codons
“Degenerate Code: Several Different Codons per Amino Acid AA”
Lecture 11-30 Transcription Translation
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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
How do we go from mRNA to Protein?
Incoming tRNA
Lecture 11-30 Transcription Translation
6
Transfer RNA
•
•
Acts as an adaptor molecule between mRNA and peptide sequence
Contains amino acid AA attachment site and template recognition site
H H
tRNA
N
N 5
O
O P O 5'
H O
O H
4'
H
9
1'
H
3'
H
O
2' H
C
O C
H
1
N
7 6
4
2
8N
N
3
+
N H
H
H
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 ()
Lecture 11-30 Transcription Translation
7
T-RNA has an L Tertiary Structure
5’
T Loop 

3’ Stem Acceptor
D Loop 
V Loop

Anti-Codon
Lecture 11-30 Transcription Translation
8
t-RNA Formation
Rnase P
Rnase P
P
5'
A
C
T C
G
GG
U
RNase D
3'
C CA
P
C CA
C
T C
G
A
GG
RNase D
U
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
N
1
N 5
t RNA
7
H
6
4
N
2
O P O 5'
8N
N
H O
3
9
O H
Charged tRNA -76 -76 4' H H 1'
3'
H
O
C
O C
H
2' H
+
+
N H
H
H
Lecture 11-30 Transcription Translation
9
tRNA Activation by aminoacyl tRNA synthases
1. Amino-acyl-AMP formation
H O
O
P
O
O
O
P
R O
O
O
H
Adenine
+ C
O
P
N
C
O
H
O
H
O
H H
O
H O
R
H
H
N
+
C
O
C
O
Adenine
P
H
O
O
H
H
H
H
O
OH
O
H O
H
O OH
2. Aminoacyl transfer to the appropriate tRNA
R
H
H
+
N CC
O
Adenine
O
P
H
O
O
O
H
O
O
H
H
HO
H
+
H O
O
P
O H
H
H
O
O H
P +
O H H
+
H
H
P
O
O
O
O
O
O
+
R
O
H
+
N CC
H
H
O
Adenine
O
O
O
O
H
P
O ACC t RNA
O
P
H
+
O ACC t RNA H
H
H
O
H
H
OH
O
OH
tRNA Synthetase Proofreading Ile
Large
Smaller
Acylation Site Hydrolytic Site
Large
Smaller
Acylation Site Hydrolytic Site
H
H
CHH
H
C
H H
H
O
O
+
N
N
H
CH
HC
H
H
H
H
O
Ile
t RNA

Difference 
t RNA
Ile
+H
H
H
O
H
H
C H H
H
C
H
H
H N
H
H
O
+
H H
H H
C
C
O
H
O
t RNA
Correct Acylation
Isol eucine
Isol eucine
t RNA O
H
+
N H
H
Mis-acylation
Lecture 11-30 Transcription Translation
10
tRNA Synthetase Proofreading Val
Hydrophobic
Acylation Site
H C
H
H
Polar
Hydrolytic Site
C H
H
H
H
H
O
C
H
H
O
O
+
N
Hydrophobic
Acylation Site
H
H
H
Polar
Hydrolytic Site
+
N H
H
V al
t RNA
O
H
H C
H
H

Difference 
t RNA
H H
CH
V al
O
H
H
C
H
O H
H
O
+
H N
H
O
O
H
+
N H
H
t RNA Valine
Valine
t RNA O
Correct Acylation
Mis-acylation
tRNA Recognition by Synthetases
"
Specific recognition of the anticodon; ex. an change the anticodon for tRNA-Trp to that for
methionine and get good acylation by tRNA-Met.
"
Stem sequences can be crucial; ex. TRNA-Ala 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.
Lecture 11-30 Transcription Translation
11
tRNA Recognition by Synthetases
 S tem and ATP S ite
t-RNA 
 Additional Recognition
Anticodon Recognition 
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 11-30 Transcription Translation
12
Codon : Anticodon
First 1st Base of Anticodon is Variable

3 2 1
t RNA- 3'-X Y Z-5' anticodon
mRNA- 5'-X’Y’Z’-3' codon
1 2 3

Third 3rd Base of Codon is Variable
tRNA Anticodon-Codon Recognition
Adenosine
H
H
N
N
N
Inosine
Guanosine
O
O
H
N
N
N
H
N
N
N
Ribose
N
H
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
¥C1 N
5’ – C – G – C – 3’
O HN H
N
NH N
N
N
O
I------C base pair
N
N
O HN
N
N
NH N
N
H
¥C1
I-----A base pair
U – 3’
O
H
H
N
O
¥C1 N
¥C1
NH O
N
¥C1 N
I----- U base pair
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
Codon
5’ – C – G –
5’ – C – G –
5’ – C – G – G – 3’
H
N
N
U – 3’
¥C1
Lecture 11-30 Transcription Translation
13
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 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.