Transcript Nucleoside Phosphoramidate Monoesters: Potential

Formation of RNA Polymerase II pre-initiation complex
IID contains TBP that binds TATA box
IIA stabilizes IID binding to promoter
IIB binds initiation sequence
Pol II binds IIB
IIE stimulates transcription
IIH has kinase and helicase activity
RNA Synthesis in E. Coli
Transcription
bubble
RNA splicing in eukaryotes
Primary transcript,
hnRNA
Alternative splicing patterns give rise to multiple proteins from
the same pre-mRNA
RNA Synthesis: Take Home Message
1) DNA sequences are translated into RNA
messages by RNA polymerases.
2) The initiation of RNA synthesis is controlled
by specific DNA promoter sequences.
3) The synthesis of RNA is governed by
initiation, elongation, and termination steps.
4) Eukaryotic mRNA is extensively processed
Overview of Protein Synthesis (Translation). Transfer
RNA.
Required reading: Stryer’ Biochemistry 5th edition Ch. 5, p. 132-136,
Ch. 28 p. 797, Ch. 29 p. 813-823
or Stryer 4th edition p. 102-104, 109-112, Ch. 34, p. 875-888 and Ch. 33 p.
849-850
Flow of Genetic Information
replication
DNA
RNA
Proteins
transcription translation
DNA
Cellular Action
How do we go from mRNA to Protein?
DNA
mRNA
Protein
t-RNA
Amino Acid
Sequence
Transfer RNA
• Acts as an adaptor molecule between mRNA and peptide sequence
• Contains amino acid attachment site and template recognition site
NH2
N
tRNA
N
O
oO
N
P
O
N
O
H
H
H
H
O
O
OH
C
CH
NH3
R
aminoacyl tRNA synthetase
Translation of mRNA
5’
N
3’
C
• mRNA is read sequentially from a fixed starting point
• Sequence of 3 Bases = One Codon (no gaps)
• One Codon = One Amino Acid
• 20 Natural Amino Acids = 64 Codons (43)
“Degenerate Code: Several Different Codons per Amino Acid”
Genetic Code
During translation, mRNA passes through the
ribosome so that each codon recognizes its tRNA
Translating the Message
DNA
5'-ATG-GCC-TTT-GAT-TCT-AAA-TAA-3'
RNA
5'-AUG-GCC-UUU-GAU-UCU-AAA-UAA-3'
Protein
N- met
ala
phe
asp
ser
Correct reading frame is essential
lys stop -C
Write the sequence of amino acids in a
polypeptide translated from the following mRNA:
5’ GGA GGA GUA AGU UGU
Gly– Gly – Val – Ser - Cys
The genetic code is nearly universal, with the
exception of mitochondria
Transfer RNA
Secondary Structure of Transfer RNA molecule
60-93 nt long
7 bp acceptor stem
O
O
H2C
H2C
NH
NH
N
O
dihydrouridine (UH2)
HN
O
pseudouridine (
Base sequence of yeast tRNAAla: cloverleaf folding result from
the presence of “self complementary” regions
-UCCGGTCGAUUCCGGA-
tRNA has an “L” Tertiary Structure
T
Loop
Acceptor
Stem
D
Loop
V Loop
Anti-Codon
Amino acid
attachment
site
Tertiary base pairs are responsible for the tertiary
structure of tRNA
#46
(m7G)
#22
G
Tertiary base
Phe
pairs in tRNA
#13
C
#46
(m7G)
#22
G
#13
C
Tertiary base
pairs in tRNAPhe
Non-standard H bond interactions, some linking 3 bases, help
stabilize the L-shaped tertiary structure of tRNA.
tRNA Formation in E. Coli
• 60 genes for tRNA are clustered in 25 transcription units
• tRNA Precursors containing several tRNAs are cleaved with RNases:
RNase P
RNase P
5'
3'
CCA
CCA
RNase D
RNase D
RNase P – generates 5’ ends of tRNA by cleaving the bond 5’ to each tRNA
RNase D – trims the 3’ end up to the CCA sequence
tRNA Activation (charging) by aminoacyl tRNA
synthetases
Aminoacyl
tRNA synthetase
Two important functions:
1.
Implement genetic code
2. Activate amino acids for
peptide bond formation
The key enzymes:
Amanoacyl-tRNA synthetases
Aminoacyl-tRNA Synthesis
Summary of 2-step reaction:
1. amino acid + ATP  aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA  aminoacyl-tRNA +
AMP
The 2-step reaction is spontaneous overall,
because concentration of PPi is kept low by its
hydrolysis, catalyzed by Pyrophosphatase.
tRNA Activation by aminoacyl tRNA synthetases
1. Aminoacyl-AMP formation:
HO O
(-)O
O
P
R
O
O(-)
O
P
C
O
O
+H 3N
O
O(-)
+H 3N
R
P
O-
Adenine
O
O
C
O
O
Adenine
O
P
O
O-
+
PPi
OH OH
Aminoacyl adenylate
(Aminoacyl-AMP)
OH OH
2Pi
2. Aminoacyl transfer to the appropriate tRNA:
R
R
O
+H 3N
C
O
O
P
O
O-
Adenine
O
+
HO-ACC-tRNA
O
+H 3N
C
ACC-tRNA
+
AMP
O
OH OH
Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi
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)
Main Differences between the two classes:
1. Structural differences. Class I are mostly monomeric,
class II are dimeric.
2. Bind to different faces of the tRNA molecule
3. While class I acylate the 2’ hydroxyl of the terminal Ado,
class II synthetases acylate the 3’-OH
Class I and II synthetases bind to different faces of the tRNA molecule
Class I synthetases
acylate the 2’-OH
Class II synthetases
acylate the 3’-OH
NH2
NH2
tRNA
N
tRNA
N
N
N
O
O
o-
oO
N
N
P
O
N
P
O
O
O
O
H
H
H
H
H
H
OH
O
O
H
O
H
O
C
C
CH
CH
R
OH
NH3
R
NH3
N
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct amino
acids.
2. tRNA synthetases recognize correct amino acids by specific
binding to the active site and proofreading.
3. tRNA synthetases recognize correct tRNAs via by interacting with
specific regions of tRNA sequence.
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct amino
acids.
2. tRNA synthetases recognize correct amino acids by specific
binding to the active site and proofreading.
3. tRNA synthetases recognize correct tRNAs via by specific
regions of tRNA sequence.
The acylation site of threonyl tRNA synthetase contains a Zinc ion
that interacts with the OH group of Threonine
O
H2N
CH C
O
OH
H2N
CH C
CH OH
CH CH3
CH3
CH3
Thr
Val
OH
Threonyl-tRNA synthetase contains editing site
O
H2N
CH C
CH OH
O
OH
H2N
CH C
H2C
CH3
Thr
Ser
OH
OH
tRNA Synthetase Proofreading
•“Double sieve” based on size
• Flexibility of the acceptor stem essential
tRNA Synthetase Proofreading
Larger
Acylation Site
Larger
Acylation Site
Smaller
Hydrolytic Site
Smaller
Hydrolytic Site
CH 3
H 3C
CH 3
CH 3
O
O
NH 3+
+H 3N
tRNAIle
O
CH 3
O
tRNAIle
Difference in Size
H 3C
CH 3
O
O
+H 3N
+H 3N
O
CH 3
O
tRNAIle
Ile
Correct Acylation
Val
Misacylation
tRNAIle
tRNAVal Synthetase Proofreading:
hydrophobic/polar recognition motif
Hydrophobic
Acylation Site
3HC
Polar
Hydrolytic Site
Hydrophobic
Acylation Site
Polar
Hydrolytic Site
CH 3
H 3C
O
OH
O
+H 3N
NH 3+
O
tRNAVal
tRNAVal
O
Difference in Hydrophobicity
CH3
CH 3
HO
CH 3
O
O
+H 3N
+H 3N
O
tRNAVal
Val
Correct Acylation
O
tRNAVal
Thr
Misacylation
The accuracy of protein synthesis depends on correct
charging of tRNAs with amino acids
1. tRNA synthetases must link tRNAs with their correct amino
acids.
2. tRNA synthetases recognize correct amino acids by specific
binding to the active site and proofreading.
3. tRNA synthetases recognize correct tRNAs via using specific
regions of the tRNA sequence.
tRNA Recognition by Synthetases
• different recognition motif depending on synthetase
• usually just a few bases are involved in recognition
•Can involve specific recognition of the anticodon
(e.g. tRNAMet), stem sequences can (e.g. tRNAAla),
both stem regions and anticodon (e.g. tRNAGln), or,
less frequently, D loop or T loop bases.
Examples of tRNA Recognition by aminoacyl
tRNA Synthetases
tRNAAla
5'P
G3
3'OH
A
C
C
tRNAPhe
5'P
tRNASer
3'OH
A
C
C
5'P
U70
C11
A
G24
D
G34
A36
A35
3'OH
A
C
C
Threonyl tRNA synthase complex with tRNA
Codon-anticodon recognition between
tRNA and mRNA
The relationship between the number of
codons, tRNAs, and synthetases
Total of 61 codons, but not 61 tRNAs!
The same tRNA can recognize more than one codon
Example:
Codon
tRNA
GCU
GCC
GCA
tRNAAla (5’-IGC-3’) alanyl tRNA synthetase
3’
Synthetase
5’
CGI
5’-GCU (C,A)-3’
anticodon
codon
Codon : Anticodon Recognition
1. The first two interactions (XY-X’Y’) obey Watson-Crick
base pairing rules.
2. The third interaction is less strict (Wobble pairing is allowed)
3 2 1
t RNA- 3'-X Y Z -5' anticodon
mRNA- 5'-X’Y’Z’-3' codon
1 2 3
The Third Base of Codon is Variable
Wobble base pairing rules
5’ anticodon base
3’ codon base
C
G
A
U
U
A or G
G
C or U
I
U, C, or A
tRNA Anticodon-Codon
Recognition
Adenosine
Guanosine
Inosine
NH 2
O
O
N
N
N
HN
N
H
N
N
N
N
HN
HN
N
H
N
Ribose
Anticodon
Codon
3'
5'
C
G
NH2
O
O
I
C
N
C-I base pair
5'
3'
3'
5'
C
G
NH2
G
C
N
C1'
C1'
N
5'
3'
3'
5'
N
A-I base pair
C
G
G
C
5'
3'
I
U
O
N
N HN
HN
I
A
O
N
N
N HN
N
C1'
G
C
NH
N
N
C1'
C1'
O
N
O HN
N
N
U-I base pair
C1'
tRNA Anticodon-Codon
Recognition
Anticodon
Codon
3'
5'
C
G
G
C
5'
3'
I
U
3'
5'
C
G
Anticodon
Codon
3'
5'
C
G
G
C
G
U
5'
Anticodon
Codon
3'
5'
C
G
G
C
U
A
5'
Anticodon
Codon
3'
5'
C
G
G
C
C
G
5'
3'
3'
3'
G
C
I
C
5'
3'
3'
5'
C
G
G
C
G
C
5'
3'
5'
C
G
G
C
U
G
5'
3'
5'
C
G
G
C
A
U
5'
3'
5'
C
G
G
C
3'
3'
3'
I
A
5'
3'
Genetic Code
Nonsense suppression
Nonsense mutations = change of codon for an aa to STOP
Usually lethal – truncated 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 GUA  CUA
Mutated tRNA recognized stop codon as Tyr and prevents
chain termination
Overview of Protein Synthesis : 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.