CHAPTER FIVE: Overview of Protein Synthesis (Translation). Transfer RNA.

Required reading: Stryer’ Biochemistry 5th edition Ch. 5, p. 132-136, Ch. 28 p. 797, Ch. 29 p. 813-823or Stryer 4th edition p. 102-104, 109-112, Ch. 34, p. 875-888 and Ch. 33 p. 849-850

I. IntroductionTranslation is the process of mRNA-directed biosynthesis of peptides. Since mRNA cannot specifically bind amino acids, transfer RNA (tRNA) molecules act as adaptors. tRNAs carry the corresponding amino acids in an activated form to the site of protein synthesis in the order specified by the mRNA sequence. Peptide bond formation is catalyzed by ribosomes, large ribonucleoprotein complexes containing catalytic RNA.

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tRNA contains both amino acid attachment site and mRNA recognition site (anticodon):

II. The Genetic CodeThe genetic code is the relationship between the sequence of bases in DNA (or RNA transcript) and the sequence of amino acids in the corresponding protein. Translating the message• Amino acids are encoded by groups of three bases. Each three bases in the sequence

(codon) specify one amino acid. • During translation, mRNA passes through the ribosome so that each codon

recognizes its corresponding tRNA. The ribosome then transfers amino acids from tRNA to the growing polypeptide chain.

Genetic code

U C A GU UUU-Phe

UUC-PheUUA-LeuUUG-Leu

UCU-SerUCC-SerUCA-SerUCG-Ser

UAU-TyrUAC-TyrUAA-TermUAG-Term

UGU-CysUGC-CysUGA-TermUGG-Trp

C CUU-LeuCUC-LeuCUA-LeuCUG-Leu

CCU-ProCCC-ProCCA-ProCCG-Pro

CAU-HisCAC-HisCAA-GlnCAG-Gln

CGU-ArgCGC-ArgCGA-ArgCGG-Arg

A AUU-IleAUC-Ile

ACU-ThrACC-Thr

AAU-AsnAAC-Asn

AGU-SerAGC-Ser

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DNA mRNA Protein

t-RNA

Amino Acid Sequence

DNA mRNA Protein

t-RNA

Amino Acid SequenceAmino Acid Sequence

O

N

NN

N

NH2

O

O

HH

HH

PO

O

tRNA

CO

CH

R

NH3

OH

o-

Aminoacyl-tRNA

AUC-IleAUG-Met

ACA-ThrACG-Thr

AAA-LysAAG-Lys

AGA-ArgAGG-Arg

G GUU-ValGUC-ValGUA-ValGUG-Val

GCU-AlaGCC-AlaGCA-AlaGCG-Ala

GAU-AspGAC-AspGAA-GluGAG-Glu

GGU-GlyGGC-GlyGGA-GlyGGG-Gly

The code is highly degenerate. There are 64 possible base triplets (4 bases in 3 possible positions, 43 = 64). 61 of the codons specify amino acids, and 3 are termination signals (UAA, UAG, UGA, "nonsense" codons). Since 61 codons code for 20 amino acids, the language is degenerate. Some amino acids are specified by as many as six different codons (Arg, Leu, Ser). Codons that specify the same amino acid are called synonyms. Degenerancy minimizes the effects of possible mutations. For example, if TTC is mutated to TTT, phenylalanine would still be incorporated into the growing polypeptide (silent mutation).

• mRNA sequence is translated from a fixed starting point (usually AUG), in the 5' 3' direction. The polypetide is formed from amino terminus to carboxy terminus:

DNA 5'-ATG-GCC- TTT-GAT- TCT-AAA-TAA-3' (CODING STRAND)RNA 5’AUG-GCC-UUU-GAU-UCU-AAA-UAA-3’Protein N-met ala phe asp ser lys stop-C

• Genetic code is nearly universal (i.e., bacteria to humans). mRNA from one organism (e.g. human) can be accurately translated by a different organism (e.g E. coli). The one exception is in mitochondria, where there are some differences. For example, UGA encodes a Termination signal for genomic transcripts, however, for human mitchondria, UGA encodes for Trp.III. Transfer RNA (tRNA)

tRNA is a hybrid of RNA and an amino acid that acts as an adaptor between the mRNA and the growing peptide sequence. tRNAs are needed because mRNA cannot directly recognize amino acids. The role of tRNAs is to carry amino acids to the site of protein synthesis in the order specified by the mRNA sequence. Each tRNA contains an amino acid attachment site (at its 3' terminus) and a template recognition site, a trinucleotide sequence which is complementary to the mRNA codon specifying the tRNA amino acid (anticodon).

A. tRNA StructureAll tRNA's have a common secondary structure that can be written in a "cloverleaf" pattern.

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• tRNA is a single chain of 60-93 nucleotides

• Unusual bases are common (e.g. 5-

methylcytidine, dihydrourracil, pseudouracil)• Non-WC base pairing in some cased (G-U)

• The 5' terminal phosphate• The base sequence at the 3' end (amino acid attachment site) is CCA

• Three loops: TC loop (thymidine-pseudouracil-cytidine), DHU loop (dihydrouracil)• Variable extra arm• Anticodon loop; 5'-pyr-pyr-XYZ-modified pur.-variable base-3'

tRNA Formation

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7 bp acceptor stem

tRNA secondary structure

tRNA tertiary structure

• L-shaped conformation• Two A-double helical segments• Acceptor stem stacks with TC stem,

and the other two helices stack to form the other arm of an “L”

• Non-standards H-bond formation involved in tertiary interactions • Anti-codon and amino acid attachment sites are far apart• CCA terminus extends out and is not conformationally restricted

tRNA's are synthesized by RNA polymerases. There are 60 genes for tRNA's from E.coli that are clustered at 25 sites, which are transcribed as multimeric precursors.

• The primary transcript is cleaved by RNase P to generate the 5' terminus. In the presence of Mg2+ , RNA of RNase P is the catalytic domain.

• The 3'-end is processed by RNase D

II. Activation of Amino Acids: Aminoacyl-tRNA SynthetasestRNAs activation involves the linking of the amino acid to its cognate tRNA to form aminoacyl-tRNA:

The linkage can be either through the 3' or through the 2' position of the ribose

Importance of amino acid activation1. Conjugation of amino acid to its tRNA establishes the genetic code.

2. Peptide bond formation in solution is highly disfavored due to the large thermodynamic barrier for this reaction. The carboxylic acid group must be

activated by conversion to a tRNA-amino acid ester, or aminoacyl-tRNA. The aminoacetyl-tRNA is referred to a being charged with the amino acid.

tRNA activationThe generation of aminoacyl-tRNA's (tRNA’s “charged” with the corresponding amino acids) is a two-step process carried out by a family of enzymes called aminoacyl-tRNA synthetases.

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RNase DRNase D

RNase P

CCACCA

RNase P

3'5'

1. The amino acid is first activated by conversion of the free carboxylic acid to aminoacyl adelylate. Although stable, the aminoacyl-AMP intermediate does not leave the enzyme.

2. The amino acid is then transferred to the appropriate tRNA:

Both reactions are catalyzed by aminoacyl tRNA synthetases.

Overall reaction:

amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi

Aminoacyl tRNA synthetase enzymes

At least one aminoacyl-tRNA synthetase exists for each amino acid. They can be grouped into Class I and Class II based on their structure and the mode of tRNA recognition.

• Class I: aminoacyl-tRNA synthetases for Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger, hydrophobic Amino Acids)

• Class II: aminoacyl-tRNA synthetases for Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr (Generally the smaller amino acids)

Differences between the two classes of tRNA synthetases

1.Structural differences. Class I are mostly monomeric, class II are dimeric.

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2 Pi

Amino acid

2.Bind to different faces of the tRNA molecule

3. While class I aatRSs acylate the 2’ hydroxyl of the terminal adenylate, Class II aatRSs

synthetases acylate the 3’-OH.

C. Accuracy and ProofreadingThe accuracy of protein synthesis depends on correct charging of tRNAs with amino acids. Aminoacyl-tRNA synthetases are highly selective for their amino acid and tRNA.

tRNA synthetases recognize correct amino acids by specific binding to the active site and proofreading.

tRNA synthetases recognize correct tRNAs via by interacting with specific regions of tRNA sequence.

1. Amino acid recognition and proofreading

Recognition of some amino acids by aminoacyl-tRNA synthetases is based on the size of the amino acid side chain. Many synthetases have a separate proofreading site that hydrolyses incorrectly charged tRNAs.

• Acylation site rejects amino acids that are larger than the correct one because the binding site is too small.

• Hydrolytic site destroys activated intermediates that are smaller than the right amino acid.

Example: Valine vs isoleucine (isoleucine has an extra methylene group)If valine is mistakenly activated by tRNA coding for isoleucine, it is hydrolyzed preventing its incorporation in tRNAIle :

• Acylation and hydrolytic sites can also discriminate based on hydrophobic verses polar interactions.

Example: Valine vs threonine (difference is –OH in place of CH3)If threonine is mistakenly activated by tRNAVal, it is rapidly hydrolyzed, preventing its incorporation in tRNAVal :

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Val is preferred Thr is preferred

• Some aminoacyl-tRNA synthetases are capable of discrimination solely at the acylation step (i.e., tyrosyl-tRNA synthetases) and do not have a hydrolytic site.

2. tRNA recognition

Recognition of the correct tRNA by the aminoacyl tRNA synthetase: challenging because of similar tertiary structure of all tRNAs different recognition motif depending on synthetase usually just a few bases are involved in recognition can involve specific recognition of the anticodon (e.g. tRNAMet, tRNAPhe), stem

sequences (e.g. tRNAAla), both stem regions and anticodon (e.g. tRNAGln), or, less frequently, D loop (tRNASer) or T loop bases.

Crystal structures demonstrate that aminoacyl tRNA synthetases interact with their tRNAs specifically at these residues.

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IV. tRNA Anticodon-Codon Recognition

To transcribe the mRNA, the tRNA's must recognize the codon. This recognition does not depend on the amino acid conjugated to the tRNA. It is based on the anticodon sequence of tRNA:

• Since the steric requirements for the third base are not stringent, less than optimal base pairing or "wobble" base pairing can occur between position 1 of the anticodon and position 3 of the codon. Recall that the genetic code is degenerate at position 3. Therefore, the base inosine, which can form wobble base pairs to U, C and A, is usually found at position 1 of the anticodon.

Inosine can base pair with C, A, or U, contributing to the degeneracy of the genetic code:

• Other wobble base pairings also can exist, e.g. G:U• The following rules apply:

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X Y ZX' Y' Z'

C G AG C U

1 2

3 2

3

15'3'3'5'

Codon

Anticodon

5' 3'3' 5'

HN

N N

N

OInosine

Ribose

1) First two bases of a codon pair in the standard way; G:C, A:U2) First base of anticodon determines whether the tRNA is able to recognize, one,

two or three codons:

This means that the same tRNA can recognize several codons specifying their amino acid.

Nonsense suppressionNonsense mutations = change of codon for an aa to STOPUsually lethal – truncated protein. Can be rescued by mutation in a different part of the genomeMechanism: tRNA gene mutation

Example: E. Coli Amber suppressortRNATyr anticodon change GUA to CUAMutated tRNA recognized stop codon as Tyr and prevents chain termination.

Protein Synthesis: Take home message

1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid. 2) The mRNA message is read by tRNA through the use of a three base complement to the three base word. 3) A specific amino acid is conjugated to a specific tRNA by aminoacyl tRNA synthetase.4) Amino acid side chain size, hydrophobicity, and polarity govern the ability of tRNA synthetases to conjugate a specific three base message with a specific amino acid. 5) Translation of RNA sequence into protein sequence takes place according to codon-anticodon interactions.

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5’ anticodon base 3’ codon base

C GA UU A or GG C or U I U, C, or A