TRANSLATION Student Edition 5/24/13 Version

Pharm. 304 Biochemistry

Fall 2014

Dr. Brad Chazotte 213 Maddox Hall

chazotte@campbell.edu

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