• • Protein synthesis “Translation”Protein synthesis “Translation”The letters of the nucleic acid is translated into amino acids.

“from nucleotide language to amino acid language”

• Genes specify the amino acids sequence in proteins.

Genetic code: the relation between the sequence of bases in DNA (or it’s transcript RNA) and the sequences of amino acid in protein.

• Features of Genetic Code

- Coding ratio (3 base-code)

- We have 4 bases and 20 amino acid:

Single-base code = 4

Two-base code = 4 * 4 = 16

Three-base code = 4 * 4 * 4 = 64

A. Three-base code

B. More than code can specify one amino acid

An amino acid is coded by three bases called “codon” and these condones:

- Non-overlapping

- The sequence of bases is read sequentially from a fixed starting point.

- There are no commas between these triplets.

• The genetic code is specificThe genetic code is specific

Specific codon always codes for the same amino acid

• • RedundantRedundant

- For a given amino acid may have more than one codon for it.

- Codons that specify the amino acid are called “synonyms” most of them differ only in the last base of the triplet

UUU

UUC

• • UniversalUniversal

The genetic code almost universal in the whole of prokaryotic, plant, animal kingdoms, the same codon used for the same amino acid.

- With few exceptions: like in the mitochondria

Codon Common code Mitochondrial code

AUC Ile Met

AGA Arg STOP

AGG Arg STOP

UGA STOP Trp

phe

Third letter

of codon

UC

AG

UC

AG

UCAGUC

AG

STARTSTART AUGAUG

STOPSTOP UAA , UAG UGAUAA , UAG UGA

• Consequences of altering the nucleotide sequence Consequences of altering the nucleotide sequence “mutation”“mutation”

A.A. Base substitution “Point mutation”Base substitution “Point mutation”

- Changing a single nucleotide base on the m-RNA chain, and this can lead to:

1. Silent mutation

The codon containing the changed base codes for the same amino acid

UCA silent UCU

Serine Serine

2. Missense

The change results in a new different amino acid

UCA missense CCA

Serine Proline

3. Non-sense mutation

The change leads to premature termination if the codon containing the changed base become a termination codon.

UCA non-sense UAA

Serine STOP codon

B.B. Base Deletion or Insertion Base Deletion or Insertion

1. Frame shift mutation

Insertion or deletion of one or two bases will alter the reading frame and this cause extensive change in the translated protein absolutely different protein

2. Insertion or deletion of one codon “3 nucleotides”

This lead to addition of new amino acids (if three bases were inserted), or to deletion of one amino acid (if three bases were deleted).

The reading frame in this case is not changed and the produced protein is not extensively changed.

Missense MutationMissense Mutation

Non-Sense MutationNon-Sense Mutation

• The Major Participants in TranslationThe Major Participants in TranslationA large number of components are required for the synthesis of

polypeptides 1. Amino acids: absence of 1 amino acid termination of the

polypeptide at that amino acid2. m-RNA: act as template for protein synthesis.3. t-RNA: adaptors4. Functional Ribosomes: protein synthesis machine.5. Energy sources6. Translation factors 7. Enzymes- The translation takes place in the cytosol

• t-RNA - At least one specific t-RNA is required for each amino acid. In

human there are 50 types of tRNA and in prokaryotes there are 30 – 40 tRNA

- 20 amino acid more than tRNA type for a given amino acid- tRNA has uncommon and modified bases (Inosine,

Pseudouracil, … )- All tRNA types have a common structure

• • tRNA structuretRNA structure

- Two functional parts

A. Acceptor stem (amino

acid attachment site)

3’-terminus of tRNA has always

the sequence 5’ … CCA-OH 3’

A. Anti codon

Three base nucleotide

sequence. That recognize a

specific codon on the mRNA

and they are complementary

and anti parallel, the codon

specifies the amino acid that

will be inserted into the

growing polypeptide.

tRNA StructuretRNA Structure

• Codon Recognition by tRNA

- Recognition of a codon in the mRNA is accomplished by anti codon sequence of the tRNA

- Some tRNA can recognize more than codon

- Anti codon + codon binding follows the complementary and anti parallel binding

• Wobble hypothesis

- The base at the 5’- end of anti codon is not spatially defined and this allows non-traditional base pairing with the 3’- base of the codon.

- The result of wobbling is that there need not be 61 tRNA types to read the 61 codons that code for the amino acidsAnti codon 3’… UAC …5’

Codon 5’ …AUG …3’

Anti codon 5’ …CAU …3’

Wobble position

•Coupling of tRNA to amino

acids

- Amino acids are covalently

attached to OH group of the

ribose sugar of the adenosine

residue at the 3’- end of tRNA.

- Each aminoacyl tRNA

synthestase recognizes a

specific amino acid and the

tRNAs that correspond to that

amino acid.

- These enzymes are highly

specific

tRNA – amino acid = activated

amino acid or charged tRNA.

• Ribosomes

Machines for protein synthesis.

- rRNA – protein complex

- Major cell constituents, an E. coli contains 15000 ribosomes forming 25% of the dried cell

- In eukaryotic cell the ribosomes either free in the cytosol or in close association with endoplasmic reticulum (ER)

- Mitochondria contains their own set of ribosomes.

• Ribosomal proteins

- These proteins play important roles in the structure and function of the ribosome.

• • The Mechanism of TranslationThe Mechanism of Translation

- The pathway of protein synthesis is called translation. Because the language of nucleotides of the mRNA is transcripted into amino acid language.

- The mRNA is translated in 5’ 3’ direction producing polypeptide from it’s amino terminal end to its carboxylic terminus.

- One prokaryotic mRNA can code for different polypeptide types (poly cistronic). Because m-RNA contains different coding regions with different initiators.

Each eukaryotic mRNA code only for one polypeptide (mono cistronic)

Code for protein A

Code for protein B

AUGAUG UAGUAA

5’

3’

•Steps in protein Steps in protein synthesissynthesis

A. Initiation

The small ribosomal subunit

Formyl group is added to the charged tRNA met by the enzyme transformylase (formyl THF is the source)

Will be Met in eukaryotes

The formyl group will be removed during the elongation

The Met amino acid will be cleaved from the polypeptide.

Specifies the next a.a

(Shine-Dalgarno sequence)

The release of IF3 increase the affinity to the large ribosomal subunit

GTP

• The binding of mRNA to 30 S ribosomal subunit

The 16S rRNA has a nucleotide sequence near it’s 3’ – end that complementary to Shine-Dalgarno sequence (nucleotide bases 5’ – UAAGGAGG – 3’ located 6 – 10 bases up stream to the AUG codon on the mRNA)

- The mRNA 5’- end and 3’- end of rRNA (in the 30S ribosomal subunit) can form complementary base pair and this can facilitate the binding of the mRNA to 30S ribosomal unit.

B. Elongation

The addition of a.a to the carboxyl end of the growing polypeptide chain.

Peptidyl transferase (integral part of 50S subunit)

The delivery of a.a - tRNA to A site GTP

Translocation: Translocation: Moves by 3 nucleotides

ElongationElongation

This process will be repeated until a termination codon is reached.

By each cycle the polypeptide has grown by one residue and consumed two GTP.

GTP

C.C. TerminationTermination

RF1 recognizes UAA and UAG

RF2 recognizes UAA and UAG

RF3 is GTPase (stimulate the release process via GTP binding and hydrolysis)

TerminatiTermination codonson codons

UAAUAA

UAGUAG

UGAUGAGTP

•Polyribosomes (polysomes)Polyribosomes (polysomes)Many ribosomes can simultaneously translate one mRNA.

• Energetic of translation

- The energy cost for protein synthesis is high.

- The total energy required for synthesizing a protein of N residues.

2N ATPs are required to charge tRNAs

1 GTP is needed for initiation.

N –1 GTPs are needed to form N –1 peptide bonds

N –1 GTPs are needed to form N –1 translocation steps

1 GTP is needed for termination

So the total energy:

2N+1 + N-1 + N-1 + 1 = 4 N

• Post translational modification. ”The final stage of protein synthesis”

Folding and covalent modification.

The produced protein may fold to form the 3° structure and may associate with other subunits.

The covalent modification involve:

Phosphorylation, Glycosylation, Hydroxylation

Trimming

Protein synthesis “Translation”Protein synthesis “Translation”

The End

GOOD LUCK

Activation of the amino acid

Aminoacyl-tRNA synthestase

Formation of ester bond

Adding the amino acid to the specific tRNA

•Coupling of tRNA to

amino acids

- Amino acids are covalently

attached to OH group of

the ribose sugar of the

adenosine residue at the 3’-

end of tRNA.

- Each aminoacyl tRNA

synthestase recognizes a

specific amino acid and the

tRNAs that correspond to that

amino acid.

- These enzymes are highly

specific

tRNA – amino acid =

activated amino acid or

charged tRNA.

• Steps in protein synthesis

A. Initiation

The small ribosomal subunit

Formyl group is added to the charged tRNA met by the enzyme transformylase (formyl THF is the source)

Will be Met in eukaryotes

The formyl group will be removed during the elongation

The Met amino acid will be cleaved from the polypeptide.

Specifies the next a,a

(Shine-Dalgarno sequence)

The release of IF3 increase the affinity to the large ribosomal subunit

The delivery of a.a - tRNA to A site

Peptidyl transferase (integral part of 50S subunit)

Moves with 3 nucleotides Translocation

By each cycle the polypeptide has grown by one residue and consumed two GTP.

This process will be repeated until a termination codon is reached.

C. Termination

RF1 recognizes UAA and UAG

RF2 recognizes UAA and UAG

RF3 is GTPase (stimulate the release process via GTP binding and hydrolysis)

UAA

UAG

UGA