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8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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 adaptorii). Amino acid activation
iii). Aminoacyl tRNA synthetases
iv). Charged tRNA
c). The genetic code
i). Codon-anticodon interactionsii). Initiation codon in prokaryotes vs. eukaryotes
iii). Reading frame
d). Mutations affecting translation
i). Frameshift mutations
ii). Missense and nonsense mutations
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Learning Objectives
Understand the structure of the ribosome and polysome
Understand the role of tRNA as an "adaptor" Understand the mechanism for charging tRNAs
Understand the general characteristics of the genetic code Know the start and stop codons Understand the mechanism for how some tRNAs can recognize more
than one codon Understand how the AUG initiation codon is recognized in prokaryotes
and eukaryotes
Understand how the AUG codon establishes the reading frame Know the various kinds of mutations that affect the translation of
proteins
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Messenger RNA (mRNA)
m7Gppp
Cap
55 untranslated region
AUG
initiationcodon
translated (coding) region
(AAAA)npoly(A)
tail
3 untranslated region
UGAtermination
codon
3AAUAAA
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Ribosomes prokaryotic ribosome
eukaryotic ribosome
70S ribosome
80S ribosome
50S subunit23S rRNA
5S rRNA
35 proteins
60S subunit28S rRNA
5S rRNA
5.8S rRNA
49 proteins
30S subunit16S rRNA
21 proteins
40S subunit18S rRNA
33 proteins
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Polysomes direction of translation is 5 to 3 along the mRNA direction of protein synthesis is N terminus to C terminus
UGA5
large ribosomal subunit
small ribosomal subunit
AUG
polysome
nascent
polypeptide
NN
subunits dissociate
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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 adenosineanticodon stem and anticodon loop
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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H2N-C-C-OH
H
R-
-
O=
ATP
H2N-C-C-O-P-O-ribose-adenine
H
R-
-
O=
amino acid
adenylated (activated)
amino acid
PPi
uncharged tRNA
H2N-C-C-OH
R-
-
O=
aminoacyl
(charged)
tRNA
AMP
3
Amino acid activation
and
tRNA charging
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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
3 additional amino acids (Arg, Leu, and Ser) are specified by
six different codons
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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The Genetic Code
UUU
UUCUUA
UUG
CUU
CUC
CUACUG
AUU
AUC
AUAAUG
GUU
GUC
GUA
GUG
UCU
UCCUCA
UCG
CCU
CCC
CCACCG
ACU
ACC
ACAACG
GCU
GCC
GCA
GCG
UAU
UACUAA
UAG
CAU
CAC
CAACAG
AAU
AAC
AAAAAG
GAU
GAC
GAA
GAG
UGU
UGCUGA
UGG
CGU
CGC
CGACGG
AGU
AGC
AGAAGG
GGU
GGC
GGA
GGG
Phe
Leu
Leu
Val
Ile
Met
Ser
Pro
Thr
Ala
Tyr
Stop
His
Gln
Asn
Lys
Asp
Glu
Cys
Arg
Ser
Arg
Gly
Stop
Trp
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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) A with U or I G with C or U U with A, G, or I I with C, U, or A
5 3
A U G
U A C
3 5 tRNAmet
mRNA
5 3
C U A
G
G A U
3 5 tRNAleu
mRNA
wobble base
one tRNAleu can read two
of the leucine codons
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Inosine = Cytidine Inosine = Adenosine
Inosine = Uridine Guanosine = Uridine
Wobble Interactions
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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
eukaryotic mRNA
lac I P O lac Z lac Y lac A
AUG AUG AUG
AUGSD AUGSDAUG
initiation codon with
Shine-Dalgarno site
initiation codon with
Shine-Dalgarno site
internal Met codon
does not have
Shine-Dalgarno site
5
5 cap AUG
initiation can only occur at
first AUG codon downstream of the 5 cap
AUG
internal (downstream) Met codon
cannot serve as an initiation site
AUG
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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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 andthe formation of a truncated protein
...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA...
MET.ALA.ASP.GLY
8/8/2019 Genetics, Lecture 6, Translation 1 (Slides)
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Mutations affecting translation hemoglobin Wayne (3 terminal frameshift mutation)
Normal -globin .ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA.THR.SER.LYS.TYR.ARG
Wayne -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