protein synthesis “translation” protein synthesis “translation” the letters of the nucleic...
TRANSCRIPT
• • 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