Translation

TRANSLATION

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Dr Mukhtiar Baig BUMDC

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Learning Objectives • Understand the general characteristics of the genetic code • Know the start and stop codons • Understand the wobble hypothesis and its importance • Understand the structure of the ribosome • Understand the role of mRNA, tRNA and rRNA in protein synthesis • Understand the steps of translation • Understand the importance of posttranslational modifications

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Books recommended for this topic

• Lehningertext book of biochemistry (excellent) • Harper’s biochemistry (good) • Lippincottsbiochemistry (good)

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The genetic code • Dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. • Each individual word in the code is composed of three nucleotide bases. • These genetic codons. 29/04/09

words

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• Codons are presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). • Nucleotide sequences always the 5′-end to the 3′-end.

from

• The four nucleotide bases produce the three-base codons. • 64 different combinations of bases. 29/04/09

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How to translate a codon: This table (or “dictionary”) can

be used to translate any codon sequence and, to determine which amino acids are coded for by an mRNA sequence.

• Eg. The codon 5′-AUG-3′ codes for methionine. • Sixty-one of the 64 codons code for the twenty common amino acids 29/04/09

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Codons With Special Roles Start Codon:

AUG Stop Codons:

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UAG BUMDC

UGA 1

Characteristics of the genetic code 1. Universality: 3. Degeneracy: 3. Specificity: 4. Nonoverlapping and nopunctuation:

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Wobble hypothesis • “The third base of the tRNA anticodon does not have to pair with a complementary codon (as do the first two) but can form base pairs with any of several related codons.”

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Wobble hypothesis

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Messenger RNA (mRNA) start codon mRNA A U G G G C U C C A U C G G C G C A U A A codon 1codon 2codon 3codon 4codon 5codon 6codon 7 gly gly ser ile ala stop protein met codon Primary structure of a protein aa1

aa2

aa3

aa4

aa5

aa6

peptide bonds 29/04/09

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tRNA

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Ribosomal Subunits

L

Large subunit

Complete functional Small ribosome subunit

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Ribosomal RNA

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Protein Synthesis Takes place in five stages • Activation of precursors • Initiation • Elongation • Termination • Folding and Posttranslational Processing

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Components Required for the Five Major Stages of Protein Synthesis in E. coli 1. Activation of amino acids • 20 amino acids • 20 aminoacyltRNA synthetases • 32 or more tRNAs • ATP • Mg+2

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2. Initiation mRNA • NFormylmethionyltRNAfmet • Initiation codon in mRNA (AUG) • 30S ribosomal subunit • 50S ribosomal subunit

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4. Termination 3. Elongation and release • Functional 70S • Termination ribosome codon in mRNA (initiation complex) • Release factors (RF-1, RF-2, RF• Aminoacyl3) tRNAs specified by codons

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5. Folding and posttranslational Specific enzymes, cofactors,

and other components for removal of initiating residues and signal sequences, • Additional proteolytic processing, • Modification of terminal residues, and • Attachment of phosphate, methyl, carboxyl, carbohydrate, or prosthetic groups •

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Stage 1: Aminoacyl-tRNA synthetases attach the correct tRNA • amino Aminoacids acidto +their tRNA + ATP

Mg2+ synthetase

Aminoacyl- tRNA

Aminoacyl-tRNA + AMP + PPi

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M • Amino acid + tRNA + ATP g2+

aminoacyl-

tRNA + AMP + 2Pi ΔG0 ≈ -29 kJ/mol

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- =

- =

Amino acid uncharged tRNA R O H2N-C-C-OH 3’ H ATP adenylated (activated) amino acid RO H2N-C-C-O-P-O-ribose-adenine PPi H

- =

RO AMP H2N-C-C-O Amino acid activation H and tRNA charging aminoacyl (charged) tRNA 29/04/09 BUMDC

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3’ end of tRNA

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Aminoacyl-tRNA synthetases

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3’ end of tRNA

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Stage 2: A Specific Amino Acid Initiates Protein Synthesis

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Formation of the initiation complex takes place in • In step 1 the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3, then mRNA binds to the 30S subunit. • In step 2 GTP-bound IF-2 and the initiating fMet-tRNAfMet are joined to the complex • In step 3 this large complex combines with the 50S ribosomal subunit.

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Protein factors required for initiation of translation in bacterial and eukaryotic cells

Bacterial

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EUKAROYTIC

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Stage 3: Elongation Stage It completes in three steps • 1. Binding of an Incoming Aminoacyl-tRNA • 2. Formation of peptide bonds • 3. Translocation

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Binding of incomin g aminoac

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Peptide bond formation

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Translocation

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Incoming aminoacyltRNA3

Direction of ribosome movementBUMDC 29/04/09

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Stage 4: Termination of Polypeptide Synthesis

Release factor binds

Polypeptidyl- tRNA link hydrolyzed 29/04/09

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Polypeptidyl-tRNA link hydrolyzed

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Components dissociate

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• •

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Stage 5: Folding and BeforePosttranslational or after folding, the

new polypeptide may undergo enzymatic processing, including Removal of one or more amino acids; Addition of acetyl, phosphoryl, methyl, carboxyl, or other groups to certain amino acid residues; Proteolytic cleavage; and/or Attachment of oligosaccharides or prosthetic groups.

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Phosphorylation dephosphorylation

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Carboxyation 29/04/09

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Loss of Signal Sequences

N + H 3 Signal sequence

C-peptide

C-peptide

N H3+ -S -S S-S -S S-

-SH HS-- SH B-chain -SH -SH HS-

COO-

N H3+ -S -S S-S -S S-

PROINSULIN

C OOINSULIN

COO-

A-chain

PREPROINSULIN

Insulin and its precursor forms 29/04/09

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Proteolytic cleavage Enterokinase

Trypsinogen

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Trypsin

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Phosphorylation

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Hydroxylation

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Biotinylated enzyme

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Farnesylated protein

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Glycosylation

BUMDC N-acety-glucosamine

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Formation of Disulfide Cross-Links

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Conversion of proinsulin BUMDC

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Inhibitors of Protein Synthesis Acting only on bacteria • Tetracycline: blocks binding of aminoacyl tRNA to A site of ribosomes. • Streptomycine: prevents the transition from translation initiation to chain elongation also causes miscoding • Chloramphenicol: bolcks the peptidyl tranferase reaction on ribosomes 29/04/09

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• Erythromycin: binds in the exit channel of ribosomes thereby inhibits elongation of the peptide chain • Rifamycin: blocks initation of RNA chains by binding to RNA polymerase (prevent RNA synthesis).

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Acting on bacteria and eukaryotes

• Puromycin: causes the premature release of nascent polypeptide chains by its addition to the growing chain end • Actinomycin D: binds to DNA and blocks the movement of RNA polymerase (prevent RNA synthesis).

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Acting on eukaryotes but not bacteria

• Cycloheximide: blocks the translocation reaction on ribosomes. • Anisomycin: bolcks the peptidyl transferase reaction on ribosomes. • α Amanitin: blocks mRNA synthesis by binding preferentially to RNA polymerase II.

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• Some of these antibiotics have deleterious effects on human mitochondria because of their resemblance with the bacteria in their sensitivity to inhibitors. .

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Tetracycli ne

Spectinom ycin

Chloramphen icol

Hygromyc Erythromy in B Streptom Streptogra cin ycin min B Large ribosomal Small ribosomal 29/04/09 BUMDC 1 subunit subunit

The Action of Antimicrobial

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Figure 20.4