Download Protein synthesis and genetic code

7. Protein Synthesis and the Genetic Code
a). Overview of translation
i). Requirements for protein synthesis
ii). messenger RNA
iii). Ribosomes and polysomes
iv). Polarity of protein synthesis
b). Transfer RNA
i). tRNA as an adaptor
ii). Amino acid activation
iii). Aminoacyl tRNA synthetases
iv). “Charged” tRNA
c). The genetic code
i). Codon-anticodon interactions
ii). Initiation codon in prokaryotes vs. eukaryotes
iii). Reading frame
d). Mutations affecting translation
i). Frameshift mutations
ii). Missense and nonsense mutations
Overview of translation
• last step in the flow of genetic information
• definition of translation
• requirements for protein synthesis
• mRNA
• ribosomes
• initiation factors
• elongation and termination factors
• GTP
• aminoacyl tRNAs
• amino acids
• aminoacyl tRNA synthetases
• ATP
Messenger RNA (mRNA)
5’
Cap
m7Gppp
5’ untranslated region
initiation
codon
AUG
translated (coding) region
UGA
3’ untranslated region
AAUAAA
termination
codon
(AAAA)n 3’
poly(A) tail
Ribosomes
• prokaryotic ribosome
50S subunit
70S ribosome
23S rRNA
5S rRNA
35 proteins
30S subunit
16S rRNA
21 proteins
• eukaryotic ribosome
60S subunit
28S rRNA
5S rRNA
5.8S rRNA
49 proteins
80S ribosome
40S subunit
18S rRNA
33 proteins
Polysomes
• direction of translation is 5’ to 3’ along the mRNA
• direction of protein synthesis is N terminus to C terminus
nascent
polypeptide
large ribosomal subunit
N
N
5’
UGA
AUG
polysome
small ribosomal subunit
subunits dissociate
Transfer RNA
• tRNA is the “adaptor” molecule in protein synthesis
• acceptor stem
• CCA-3’ terminus to which amino acid is coupled
• carries amino acid on terminal adenosine
•anticodon stem and anticodon loop
Amino acid activation and aminoacyl tRNA synthetases
• aminoacyl tRNA synthetases are the enzymes that “charge” the tRNAs
• 20 amino acids
• one aminoacyl tRNA synthetase for each amino acid
• can be several different “isoacceptor” tRNAs for each amino acid
• all isoacceptor tRNAs for an amino acid use the same synthetase
• each aminoacyl tRNA synthetase binds
• amino acid
• ATP
• isoacceptor tRNAs
amino acid
uncharged tRNA
- =
RO
H2N-C-C-OH
3’
H
ATP
- =
H
AMP
Amino acid activation
and
tRNA charging
RO
H2N-C-C-O
- =
PPi
adenylated (activated)
amino acid
RO
H2N-C-C-O-P-O-ribose-adenine
H
aminoacyl
(charged)
tRNA
The genetic code
• consists of 64 triplet codons (A, G, C, U) 43 = 64
• all codons are used in protein synthesis
• 20 amino acids
• 3 termination (stop) codons: UAA, UAG, UGA
• AUG (methionine) is the start codon (also used internally)
• multiple codons for a single amino acid = degeneracy
• 5 amino acids are specified by the first two nucleotides only
The Genetic Code
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Phe
Leu
Leu
Ile
Met
Val
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Pro
Thr
Ala
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
Tyr
Stop
His
Gln
Asn
Lys
Asp
Glu
UGU
Cys
UGC
UGA Stop
UGG Trp
CGU
CGC
Arg
CGA
CGG
AGU
Ser
AGC
AGA
Arg
AGG
GGU
GGC
Gly
GGA
GGG
Codon-anticodon interactions
• codon-anticodon base-pairing is antiparallel
• the third position in the codon is frequently degenerate
• one tRNA can interact with more than one codon (therefore 50 tRNAs)
• wobble rules
• C with G or I (inosine)
3’
5’ tRNAmet
• A with U or I
• G with C or U
• U with A, G, or I
• I with C, U, or A
UAC
AUG
5’
mRNA
3’
3’
5’
• one tRNAleu can read two
of the leucine codons
5’
mRNA
GAU
CUA
G
tRNAleu
wobble base
3’
Wobble Interactions
Inosine = Cytidine
Inosine = Adenosine
Inosine = Uridine
Guanosine = Uridine
Initiation in prokaryotes and eukaryotes
• initiation can occur at internal AUG codons in prokaryotic mRNA
• initiation in eukaryotes occurs only at the first AUG codon
• lac operon in E. coli is transcribed as a polycistronic mRNA
with multiple AUG codons
lac I
P
O
lac Z
AUG
5’
SD
AUG
AUG
initiation can only occur at
first AUG codon downstream of the 5’ cap
AUG
SD
internal Met codon
does not have
Shine-Dalgarno site
AUG
lac A
AUG
AUG
initiation codon with
Shine-Dalgarno site
• eukaryotic mRNA
5’ cap
lac Y
AUG
initiation codon with
Shine-Dalgarno site
AUG
internal (downstream) Met codon
cannot serve as an initiation site
Reading frame
• reading frame is determined by the AUG initiation codon
• every subsequent triplet is read as a codon until reaching a stop codon
...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...
MET.ALA.GLU.TRP.LEU.SER.MET.SER
• a frameshift mutation
...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...
• the new reading frame results in the wrong amino acid sequence and
the formation of a truncated protein
...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA...
MET.ALA.ASP.GLY
Mutations affecting translation
• hemoglobin Wayne (3’ terminal frameshift mutation)
Normal a-globin
.ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA
.THR.SER.LYS.TYR.ARG
Wayne a-globin
.ACG.UCA.AAU.ACC.GUU.AAG.CUG.GAG.CCU.CGG.UAG
.THR.SER.ASN.THR.VAL.LYS.LEU.GLU.PRO.ARG
mutated region
• missense mutations (e.g., AGC Ser to AGA Arg)
• nonsense mutations (e.g., UGG Trp to UGA Stop)
• read through, reverse terminator, or sense mutations
(e.g., UAA Stop to CAA Gln) as in hemoglobin Constant Spring
• silent mutations (e.g., CUA Leu to CUG Leu) do not affect translation