Chapter 12
12.1 Translating the Genetic Message
tRNAs: adapter function in translation
bind specific amino acids and recognize a specific sequence in mRNA
interact with ribosome and aminoacyl-tRNA synthetases
charged with specific amino acid by specific aminoacyl tRNA synthetase
ribosome: made of many proteins and RNA molecules, this is where translation occurs
requires additional protein factors for initiation, elongation, and termination of translation
additional enzymes for modifications
12.2 The Genetic Code
triplet (codon): sequence of three bases to specify one amino acid
non-overlapping: consecutive codons do not share bases
comma-less: no intervening bases between codons
degenerate: more than one triplet codes for the same amino acid
not uniform or imperfect
unambiguous: no single codon codes for more than one amino acid
universal: same in viruses, prokaryotes, and eukaryotes
ribosome moves along the mRNA three bases at a time
64 codons
61 code for amino acids
3 (UAA, UAG, UGA) stop codons
Trp and Met have one codon each
Third base irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, Arg
Second base is important for type of amino acid
Second base is U = hydrophobic amino acid
18 amino acids coded for by multiple codons
only the third letter varies in the multiple codons
codons may have specialized functions
AUG = start codon
UAA, UAG, UGA = stop codon
tRNA: small, single-stranded RNA molecules folded into 3D structure
73-93 nucleotides long in prokaryotes and eukaryotes
3D structure allows binding to mRNA, the ribosome, and aminoacyl-tRNA synthetases
Codon-Anticodon Recognition
coding sequence is complementary/antiparallel with anticodon sequence
codon in mRNA base pairs with anticodon via hydrogen bonding
Wobble position: third base in codon, first base in anticodon
some tRNAs bind to one codon only, but may recognize more than one codon because of variations in patterns of hydrogen bonding
Watson-Crick base pairing is broken
first two bases of codon form strong Watson-Crick base pairs
base at Wobble position determine number of codons read
codons that differ in first or second base requires different tRNAs
32 tRNA required to translate 61 codons
Minimizes damage caused by misreading of code
Balance between accuracy and speed
loose pairing allows for rapid dissociation of tRNA
Protein synthesis overview
Activation of amino acids: enzymatic synthesis of aminoacyl-tRNA molecules
Initiation: binding of mRNA and N-formylmethionine to the ribosome
Elongation: binding of the aminoacyl-tRNAs to the ribosom and formation of the peptide bonds
Termination: termination codon in the mRNA is positioned in the ribosome
Folding and post-translational processing
12.3 Amino Acid Activation
two steps catalyzed by aminoacyl-tRNA synthetase
selectivity at amino acid and tRNA
amino acid: aminoacyl-AMP remains bound to enzyme and binding of the correct amino acid is verified by editing site in tRNA synthetase
tRNA: specific binding sites recognized by aminoacyl-tRNA-synthetases
occurs in cytosol
involves covalent attachment of proper amino acid to specific tRNA
free energy of hydrolysis of ATP provides energy for bond formation
amino acid covalently linked to tRNA by formation of ester between carboxylate and 2’ or 3’ OH of the 3’-adenylate of the tRNA
second genetic code: matching each amino acid with correct tRNA
molecular recognition of specific tRNA molecule by specific synthetase
only few nucleotides in tRNA confer the binding specificity
anticodon and GU in Ala-tRNA
Ribosome: cellular machine responsible for protein synthesis
non-covalent assembly of many proteins and few RNA molecules
bacterial ribosomes: two subunits of unequal size
50S: 36 proteins, 55 rRNA, 23S rRNA
30S: 21 proteins, 16S rRNA
three binding sites (A, P, E)
Aminoacyl site: incoming aminoacyl-tRNAs bind during elongation
Peptidyl site: initiating AUG codon is positioned
fMet-tRNAMet binds
Exit site: uncharged tRNA leaves after formation of peptide bond
12.4 Prokaryotic Translation
Chain initiation: initiation methionine
one methionine codon (AUG) but two tRNAs for methionine
tRNAs have same anticodon but different sequences
one used to initiate protein synthesis
one when methionine is added at internal positions within a protein
transformylase is selective - will not formylate free Met or Met-tRNAmet
N-formylmethionine = fMet = initiator tRNA
produced in two steps with Met-tRNA synthetase and transformylase
Chain initiation: components
synthesis of polypeptide chain starts at N-terminus and grows to C-terminus
initiation requires: tRNAfmet, initiation codon (AUG) of mRNA, 30S subunit, 50S subunit, initiation factors (IF-1, IF-2, IF-3), GTP, Mg2+
forms initiation complex
Step 1
30S forms complex with IF-1 and IF-3
prevents premature interaction of 30S and 50S subunits to form inactive 70S ribosome devoid of mRNA
mRNA binds to IF-1/IF-3/30S complex
IF-1/IF-3/30S subunit/mRNA complex
Shine-Dalgarno squence binds with 16S rRNA of 30S
Step 2
IF-2 GTP and fMet-tRNAfMet bind to IF-1/IF-3/30S subunit/mRNA complex
IF-2 GTP/fMet-tRNAfMet/IF-1/IF-3/30S subunit/mRNA complex
IF-2: G protein; binds to GTP enabling binding of fMet-tRNAfMet
Step 3
50S binds to IF-2 GTP/fMet-tRNAfMet/IF-1/IF-3/30S subunit/mRNA complex with hydrolysis of GTP bound to IF-2
IF-1, IF-2, and IF-3 dissociate
fMet-tRNAfMet/70S subunit/mRNA complex
overview
mRNA guided to 30S via Shine-Dalgarno in mRNA
fMet-tRNAfMet binds to P site
50S combines with 30S
A site is unoccupied, waiting for aminoacyl-tRNA to bind
Chain elongation
uses three binding sites (APE) on 50S subunit
requires 70S, codons of mRNA, aminoacyl-tRNAs, elongation factors, GTP, Mg2+
EF-Tu: elongation factor - temperature unstable
EF-Ts: elongation factor - temperature stable
EF-G: elongation factor - GTP
Elongation Step 1
aminoacyl-tRNA is delivered and bound to A site
P site is occupied
incoming tRNA binds to EF-Tu GTP complex
aminoacyl-tRNA^AA/EF-Tu GTP
bound at A site
GTP hydrolyzed releasing EF-Tu GDP from A site
Elongation Step 2
peptide bond is formed
tRNA at P site is uncharged
Nucleophilic attack of a-amino group of amino acid in the A site at the carboxylate carbonyl of the amino acid in the P site
uncharged tRNA in P site and dipeptide in A site
N-terminus to C-terminus
Elongation Step 3
uncharged tRNA is released
peptidyl-tRNA is translocated to P site
EF-G and GTP required
next aminoacyl-tRNA delivered to empty A site
ribosome moves the distance of one codon toward 3’-end of mRNA
dipeptidyl-tRNA shifts from A site to P site
uncharged tRNA shifts to E site and is released to the cytosol
next aminoacyl-tRNA binds to A site
requires EF-G and GTP hydrolysis
conformational change in ribosome
Elongation Step 4
continued formation of polypeptide
P site has dipeptidyl-tRNA and A site has charged tRNA, process continues to form more peptide bonds
ribosome moves from codon to codon toward 3’-end of mRNA
Each peptide bond requires hydrolysis of 2 GTPs
one is hydrolyzed to deliver aminoacyl-tRNA^AA to A site
one is hydrolyzed to translocate the ribosome
Termination
P site has protein-tRNA and A site has stop codon
bacteria: release factors (RF-1, RF-2, RF-3) are required
RF-1 or RF-2 bind to A site, recognizing stop codon
contribute to hydrolysis of protein from final protein-tRNA, release free protein/last tRNA/mRNA, and disassemble ribosome
summary of protein synthesis
intiation: binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit
elongation: ribosome moves along mRNA to synthesize peptide bonds with tRNAs bound to A and P sites
termination: occurs when stop codon is reached
Protein synthesis energy (4 high energy bonds are cleaved)
2 ATP required to esterify each amino acid to tRNA
1 GTP to deliver aminoacyl-tRNA to A site
1 GTP to translocate ribosome
extra energy in termination and proof-reading
Protein synthesis inhibitors
Tetracycline: binds to A site, prevents binding of charged tRNAs
Chloramphenicol: inhibits peptidyl transfer
Cyloheximide: inhibits peptidyl transfer
Streptomycin: causes mis-reading of genetic code
Puromycin: causes premature termination
Ricin: inactivates 60S of ribosome by depurinating A residue in 23S rRNA
Diphtheria toxin: inactivates EF2 by attaching ADP-ribose to amino acid
Polypeptide maturation
primary translation product must mature to form a biologically active protein
proper folding into active 3D structure
post-translational modifications
12.6 Post-translational Modification of Proteins
N- and C-terminus modifications
loss of N-terminal fMet
N-terminal acetylation
N-terminal lipidation
C-terminal amidation
loss of signal sequence
modification of amino acids
formation of disulfide bonds
attachment of carbohydrates, ubiquitin (controlled protein degradation), or prosthetic group
isoprenylation
proteolytic processing
12.7 Controlled Protein Degradation