translation student edition 5/24/13 version pharm. 304 biochemistry fall 2014 dr. brad chazotte 213...
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TRANSLATION Student Edition 5/24/13 Version
Pharm. 304 Biochemistry
Fall 2014
Dr. Brad Chazotte 213 Maddox Hall
Web Site: http://www.campbell.edu/faculty/chazotte
Original material only ©2007-14 B. Chazotte
Goals
• Understand how the structure of tRNA functions in translation
• Understand the importance of aminoacyl tRNA synthetases
• Examine how tRNAs and mRNAs interact specifically
• Understand the role of the ribosome structure/function in protein synthesis
• Learn the differences in prokaryote & eukaryote translation
• Examine posttranslation modification of proteins
Translating the Genetic Code
• An amino acid is specified by three consecutive bases called a Codon
• More than one triplet (of the 64) can specify the same amino acid.
• (the code is degenerate).
• The code is read in a sequential manner starting from a fixed point in a gene (“the reading frame”). No other guide other than reading
triplet after triplet.
• There are two INITIATION codons: AUG (most frequent) & GUG
• There are three STOP codons (of the 64): UAG, UAA & UGA
• (Mitochondria have a slightly different coding.)
Voet, Voet & Pratt 2013 Fig 27.2
Reading Frame & Translation
mRNA is read in sequence triplet after triplet
A missing base changes everything after it.
Voet, Voet & Pratt 2013 Table 27.1
The Genetic CodeCode is DEGENERATE
Codons that specify the same amino acid are called synonyms
Table arrangement seen to be NONRANDOM, e.g. most synonyms occupy same “box”.
1st position changes - tend to specify similar or same AA
2nd position changes - pyrimidines code mainly hydrophobic AA; purines mainly polar AA.
3rd position “wobble” has some conformation flexibility
AUG, GUG initiation
UAG, UAA, & UGA stop
tRNAs
• Carry an amino acid to a ribosome for protein synthesis.
• Anticodon complementary to mRNA codon makes for specific binding
• Functions to help with proofreading for translation.
Most tRNA’s ~ 76 nucleotides (54 – 100 range)
15 invariant positions
8 semivariant positions
Approximately 25% of its bases are modified
Amino acids binds to 3-OH of CCA sequence
Transfer RNA (tRNA) are “adapter” molecules that faithfully translate the information in mRNA into a specific sequence of amino acids.
Voet, Voet & Pratt 2013 Fig 27.5
Yeast tRNA
tRNA 2º and 3º Structure
Aminoacyl tRNA Synthetases
Voet, Voet & Pratt 2013 Table 27.2
Each enzyme carries out attachment of a specific amino acid to the appropriate tRNA in a two reaction sequence.
1. Activation by reaction with ATP to form aminoacyl-adenylate
2. That mixed anhydride reacts with tRNA to form aminoacyl tRNA
Two structurally unrelated enzyme classes that differ in:
• mechanism by which they recognize tRNA
• initial site of aminoacylation on the tRNA
• amino acid specificity
Voet, Voet & Pratt 2006 Fig 26.7; 2013 27.7
tRNA Structural Features are Recognized by Aminoacyl-tRNA Synthetases
Voet, Voet & Pratt 2013 Fig 27.8
Most contact sites on the tRNA by the synthetase are on the concave inner face of the L and the acceptor stem.
Other sites involved in recognition – could be a small as a
single base. Can be at opposite ends of tRNA
tRNA tRNA Synthetase
Aminoacyl tRNA Synthetase Proofreading
Thermodynamic basis If the difference in binding of similar structures differing by a methylene group is ~12 kJ mol -1
then the ratio f of the equilibrium constants, K1 and K2, with which two substances bind to a given binding site is given by the above equation where is the difference between the free energies of binding of the two substances. It is therefore estimated that isoleucyl–tRNA synthetase could discriminate between isoleucine and valine by no more than a factor of ~100. The discrimination is, in fact, much greater 3,000 to 1and is attributed to using two successive steps.
Rule of Thumb: “If available binding interactions do not provide the necessary discrimination between two substrates, binding in two successive steps provides a multiplicative filtering effect”
Some aminoacyl-tRNA synthetases use two active (binding) sites an aminoacylation
site and an editing site (hydrolyzing the aminoacylated tRNA., e.g. IleRS.
K1 e-G1º’/RT
K2 e-G2º’/RT = = e-Gº’/RT
Voet, Voet, & Pratt 2012 Chap 27 p. 973
mRNAs
• 5’ end of a mature mRNA codes the amino terminus of a protein
• mRNA codon makes for specific binding of tRNA complementary anticodon.
Messenger RNA (mRNA) are intermediates that carry the genetic information from one to a few genes to a ribosome. The template for protein synthesis
RibosomesLarge ribonucleoprotein complexes - 2.5 to 4.5 x 106 D
Functions:
“Binds mRNA such that it codons can be read with high fidelity”
“Includes specific binding sites for tRNA molecules.”
“Mediates interactions of nonribosomal protein factors that promote polypeptide chain initiation, elongation, and termination.”
“Catalyzes peptide bond formation”
“Undergoes movement so that it can translate sequential codons.”
Prokaryote and Eukaryote ribosomes carry out the same functions and their structures are similar, but the details are different. Eukaryotic ones are larger and more complex.
Voet, Voet & Pratt 2013 p.977
Voet, Voet & Pratt 2013 Table 27.4
Prokaryotic RibosomeTwo major (large) subunits that together form 70S ribosome.
50S – mainly mediates biochemical tasks, e.g., polypeptide elongation catalysis
30S – major function recognition process, e.g., tRNA & mRNA binding
The ribosome has a complicated tertiary structure.
The rRNAs in the subunits have a complicated secondary structure.
Berg, Tymoczko & Stryer 2012 Fig. 30.14
23S rRNA
proteins
16S rRNAproteins
5S rRNA
Voet, Voet & Pratt 2013 Fig 27.12
E. Coli rRNA Secondary Structure• There are a number of domains• There are many short duplex regions of the rRNA with
Watson-Crick base pairings.• There are regions of non Watson-Crick base pairings
In 30S subunit In 50S subunit
Ribosome’s Three tRNA Binding SitesOn the 30S ribosome subunit codon (mRNA)-anticodon binding:
A (aminoacyl) Site – where the “new” aminoacyl tRNA binds.
P (peptidyl) Site – where the tRNA attached to the growing peptidyl chain is attached. Peptide bond formed between polypeptide chain and new amino acid.
E (exit) Site –where the deacylated (empty) tRNA that is about to exit the ribosome is attached.
Berg, Tymoczko & Stryer 2012 Fig. 30.16 Voet, Voet & Pratt 2013 Fig 27.17
Voet, Voet & Pratt 2008 Fig 27.13b
A Ribosome Translating a mRNA
Voet, Voet, & Pratt 2013 Fig. 27.13
Voet, Voet & Pratt 2013 Table 27.5
Eukaryotic Ribosome
Eukaryotic Ribosome is larger: 60S & 40S.
18S rRNA homologous to 16S.
5S and 28S rRNA counterpart to 5S & 23S
Has unique 5.8S rRNA.
Lehninger (Nelson & Cox) 2005 Fig 27.9d
Translation
The process by which a sequence of mRNA nucleotides are copied (translated) to protein.• Proceeds from N-terminus to C-terminus• Elongation by linking growing polypeptide to incoming
amino acid on tRNA.• mRNA read 5’ to 3’ by ribosome• Active translation occurs on polysomes
Major difference between Prokaryotes and Eukaryotes:Eukaryote
Larger ribosome Met is the initiating amino acid instead of fMet but a special tRNA still used: Met-tRNAi Initiating codon always AUG Use more initiating factors Use single release factor instead of two
Sequence for the Translation Process Initiation, Elongation, & Termination
Get to the work site – (eukaryotes) mRNA transported from nucleus to cytoplasm.
Get the mRNA set – mRNA loaded on the ribosome for translation.Copy the RNA→Protein – Ribosome “reads” the mRNA, specific
tRNA binds to complementary sequence on the mRNA synthesizes RNA.Know when to stop - Ribosome comes to termination
site (stop codon & release factors involved)Goodbye - Protein separates from ribosome.Modify Protein – some proteins may undergo posttranslational
modification.
Initiation
Voet, Voet & Pratt 2013 Fig 27.25
E. Coli
(Prokaryote)
1. Inactive 70S ribosome (from previous cycle) dissociates upon IF-3 binding to 30S subunit
2. mRNA & IF-2 with GTP and fMet-tRNAf
Met bind to 30S subunit forming initiation complex.
3. IF-1 and IF-3 are released permitting the 50S subunit to join the initiation complex. This causes IF-2 hydrolyzes its GTP, causing a conformational change in 30S subunit releasing IF-2.
Voet, Voet & Pratt 2013 Chap. 27 p.986
Voet, Voet & Pratt 2013 Fig 27.23
E. Coli Translation Recognition Sequences(w/ Shine-Delgarno Sequences)
Shine-Delgarno (red): Purine-rich sequence centered ~10 nt upstream of start codon in prokaryote mRNA. Partially complementary to pyrimidine-rich sequence at 3’ end of 16S rRNA (green).
Differences in Eukaryotic Initiation
Lehninger (Nelson & Cox) 2005 Fig 27.22
More complicated and more factors involved
No fMet rather there is an initiator Met-tRNAi
No Shine-Delgarno Sequence
Eukaryotic mRNA’s cap and poly(A) tail are bound via factors to the ribosome 40S subunit.
The ribosome scans the mRNA to find the first AUG (still codes for a methionine)
The AUG is found in the consensus sequence (GCCRCCAUGG), tells ribosome this is where to start with initial methionine.
Elongation
• Decoding – selection and binding of an aminoacyl tRNA.
• Transpeptidation – peptide bond formation, peptidyl group in P-site tRNA is transferred to the aminoacyl group in
the A site.
• Translocation – A-site and P-site tRNAs are transferred to the P and E sites, respectively, and this moves the base-
paired mRNA through the ribosome the length of one codon.
Polypeptide chains are elongated by the ribosome in a three-stage cycle.
Peptide bond is formed during elongation process.
Voet, Voet & Pratt 2013 Fig 27.27
In prokaryotes: elongation factors EF-Tu, EF-Ts, & EF-G
Lehninger (Nelson & Cox) 2005 Fig 27.23
Elongation Step 1 – DecodingBind 2nd Amino Acid
P Site is occupied with an aminoacyl tRNA, (fMet-tRNAf
Met if first residue).
A-site is empty
Appropriate incoming aminoacyl-tRNA binds to EF-Tu and GTP complex
The aminoacyl-tRNA-EF-Tu-GTP complex binds to the A SITE
GTP is hydrolyzed and EF-Tu and GDP complex is released
Lehninger (Nelson & Cox) 2005 Fig 27.24
Elongation Step 2 - TranspeptidationPeptide Bond Formation
23S rRNA functions as peptidyl transferase
N-formylmethionyl group is transferred to the amino group of the second amino-acyl tRNA in the A-SITE.
Both tRNA shift position in the 50S subunit.
The (now) uncharged tRNA shifts to the E-site.
The peptidyl tRNA 3’ & 5’ ends shift to the P-site
The anticodons REMAIN in the A and P sites
Lehninger (Nelson & Cox) 2005 Fig 27.25
Elongation Step 3 - Translocation
The ribosome moves one codon toward the mRNA 3’ end using GTP hydrolysis for energy (drives ribosome conformation change).
The dipeptidyl-tRNA is now completely in the P-site. (The A-site is now open.)
Now set for new aminoacyl-tRNA to enter the A site.
Ribosomal Proofreading• Insure fidelity of translation
• Have proofreading step independent of initial selection – screening effects are multiplicative.
• Involves EF-Tu hydrolysis step
• Formation of correct codon-anticodon pair triggers GTP hydrolysis on EF-Tu and dissociation from aa-tRNA.
Termination
Utilizes Release factors RF-1 RF-2, and RF-3 (Prokarytote)
eFR1 (Eukaryote)
RF-1 recognizes UAA and UAG Stop codons,
RF-2 recognizes UAA and UGA Stop codons
(There are no tRNAs for these codons.)
Release factor induces transfer of the peptidyl group to water instead of a new aa-tRNA. Free polypeptide dissociates from ribosome.
In subsequent step other releases occur such as mRNA
Lehninger (Nelson & Cox) 2005 Fig 27.26
Posttranslational Processing
Protein Folding – during or after synthesis the nascent protein progressively assumes it native (biologically active) conformation via H-bonds, van der Waals, ionic, and hydrophobic interactions. Formation of disulfide bonds
Covalent Modifications
Phosphorylation (reversible)
Glycosylation - add carbohydrate groups
Add isoprenyl groups
Add prosthetic groups – e.g. heme groups
Proteolysis – shorten to active form
Lehninger (Nelson & Cox) 2005 Fig 27.34
Berg, Tymoczko & Stryer 2012 Table. 30.4.
Many Antibiotics Function by Inhibiting Protein Synthesis
Voet, Voet, & Pratt 2013 Box 27.3
End of Lectures