TRANSLATION

m-RNA
mRNA have a Ribosomes binding site Also known as Shine Dalgarno sequence (it is an

initiation signal of purine residues on the 5`side and bp with a complementary pyrimidine rich sequence near the 3`end of 30s ribosomal subunit) generally located 8 basepairs upstream of the start codon AUG . Coding sequence for protein and recognition elements for initiation and termination of translation. The coding sequence is known as ORF consist of 3nucleotide ORF start with start codon (AUG & GUG) and with stop codons(UAA,UAG,UGA) POLYCISTRONIC

Ribosomes

location of protein synthesis A ribosome is composed of two subunits, a large one and a small one. Prokaryotic Eukaryotic S 16S rRNA+21Ps 18S rRNA+33Ps 30S 40S L 23S rRNA 28S rRNA 5S rRNA 5.8S rRNA +36Ps 5 SrRNA +49Ps 50S 60S Whole 70S 80S Function of Ribosome To hold mRNA, aminoacyl-tRNA and translation factors in right place for protein synthesis. To catalyze certain chemical reactions in the process of protein synthesis.

t-RNA
Robert w. holley gave the clover leaf structure of t-RNA

There are 20 amino acids.

There are 64 codons out of which 61are sense codons. t-RNA help in translating the language of nucleic acids into the

language of proteins These are small and consist of ssRNA folded into 3D structure. 32 t-RNA are required to recognize all amino acid codons Yeast alanine t-RNA , the first nucleic acid to be completely sequenced Because of wobble base-pairing caused codon degeneracy 61 sense codons can be red by less than 61 tRNAs. Physical interface between mRNA and aminoacids. 3` CCA site of attachment of aminoacid 5`polyguanylate residue D ARM contain unusual nucleotide dihydrouridine

GENETIC CODE
The genetic code is the way in which the nucleotide sequence in mRNA ( or DNA ) specifies the amino acid sequence in protein. A, G, U and C are organized into triple-nucleotides called codons.
The collection of the 64 codons makes up the genetic code. The genetic code was deciphered in 1966 by Nirenberg et al. In the genetic code 61 codons specify 20 amino acids. They are sense codons. There are 3 codons, UAA, UAG and UGA,which do not specify

any amino acids. They are stop codons ( termination codons,nonsense codons ).

FEATURES OF GENETIC CODE

1. Universal The genetic code is used by all species, from prokaryotes to human being. However some deviations are known to occur in mitochondria and some unicellular organisms. 2. Directional The sequence of triple-nucleotide codons is read in the direction of 5 to 3. The first codon of coding region of mRNA is almost always AUG, the initiation codon. The last codon of the coding region of mRNA is one of the stop codons.

3. Commaless The coding region in mRNA is read in a continuing way without punctuation.

If there is insertion or deletion of one or two nucleotide(s) in the coding region of mRNA frameshift mutation occurs.

4. Degeneracy Because 61 codons specify 20 amino acids multiple codons must decode the same amino acid. That is called degeneracy. For example Leu, Ser, and Arg each is specified by six different codons.

5. Wobble

There is wobble in the process of codon( in mRNA ) and anticodon ( in tRNA ) base-pairing. That is the base-pairing does not strictly according to the standard base-pairing rule ( A-U , G-C , T-A ).

PROTEIN BIOSYNTHESIS
Protein biosynthesis takes place in 5 stages: 1) Activation of amino acids 2) Initiation 3) Elongation 4) Termination and release 5) Folding and post-translational processing

COMPONENTS
m RNA

t RNA
Ribosomes Amino acyl t-RNA synthetases ATP and GTP Inorganic ions

ACTIVATION OF AMINO ACID

Only L aa takes part (Not citrulline,alanine) Aminoacylation: tRNAs are joined to amino acids to become aminoacyl-tRNA `in presence of Special enzymes called aminoacyl-tRNA synthetases The aminoacylation reaction is a two-step reaction driven by ATP. first step : activation of amino acid. AA + ATP ------------- AA-AMP + Ppi second step : charging tRNA (specific). tRNA + AA-AMP ---- AA-tRNA+AMP tRNA becomes attached to the aminoacyl group through an ester bond.

INITIATION
Initiation requires : 30s ribosomal subunit 50s ribosomal subunit m RNA coding for polypeptide Initiating fmet-t RNA Initiation factors: IF-1: prevent premature binding of t-RNA to A site. IF-2: binding of fmet t-RNA to 30s ribosomal subunit IF-3: binds to 30s subunit, prevents premature association of 50s subunit GTP Magnesium ion

Step1: IF-1 and IF3 bind to 30s ribosomal subunit m RNA binds to 30s subunit Initiating 5`AUG is guided to its correct position by shine dalgarno sequence Bacterial ribosomes have 3 sites A (aminoacyl) site, P (peptidyl)site and E(exit) site

 Both A and P sites contribute to both 30s and 50s

subunit whereas E site confined to only 50s subunit 5`AUG is positioned at only P site to which fmet- t RNA can bind while all other amino acids binds first to A site then P site and then E site E site is that site from where uncharged t RNA leave during elongation

Step2: GTP bound IF2 and fmet t-RNA bind to the initiation complex Anticodon of t RNA now correctly pairs with m RNA initiation codon Step3: complex join to 50s ribosomal subunit
GTP GDP+ PI Initiation factors are released from the cmplex

ELONGATION
Elongation requires : Initiation complex Aminoacyl t RNA Elongation factors (EF-Tu, EF-Ts and EF-G) GTP

STEPS
There are 3 steps involved: 1. Binding of an incoming aminoacyl t RNA 2 Peptide bond formation Three sites EPA on the ribosomes
A site: receives all incoming charged tRNA P Site : previous tRNA with the new polypeptide

(fMeth tRNA directly bind) E Site: deacylation

3 Translocation

Binding of incoming acyl t-RNA

Incoming amino acyl t-RNA binds to a complex of

GTP bound EF-Tu Amino acyl t RNA-EF Tu complex binds to the A site of the 70s initiation complex GTP is hydrolysed and EF-TU GDP complex is released and is regenerated involving EF-Ts

peptide bond formation

A peptide bond is formed between the two amino

acids bound by their t RNA to the A and P sites This occurs by transfer of fmet from its t- RNA to the amino group of second amino acid now in the A site A dipeptidyl t-RNA is produced and deacylated tRNA remain bound to the p site

TRANSLOCATION
The ribosome moves one codon towards the 3`

end of m-RNA This movement shifts the anticodon of the dipeptidyl t RNA which is still attached to the second codon of the m-RNA from the A site to the P site and shifts the deacylated t-RNA from the P to the E site The t-RNA is released into the cytosol

 Third codon of the m-RNA lies in the A site while

second in the P site Movement of the ribosome along the m-RNA requires EF-G (translocase) and energy is provided by hydrolysis of GTP

TERMINATION
Elongation continues until the ribosome adds the

last amino acid coded by the m-RNA It is signaled by the presence of one of the termination codons in the m-RNA (UAA,UAG,UGA) There are 3 termination or release factors: RF1,RF2,RF3 RF1 recognises the termination codon UAG and UAA

 RF2 recognises UGA and UAA These release factor help in: 1. Hydrolysis of the terminal t-RNA bond 2. Release of free polypeptide and the last t-RNA 3. Dissociation of the 70s ribosome into 30s and

50s subunit These RF factors bind to the termination codon and induces peptidyl transferase to transfer the growing polpeptide to water molecule rather than to amino acid

POST TRANSLATIONAL MODIFICATION

In order to achieve biologically active form the

new polypeptide must fold into its proper 3D conformation Before or after folding the new polypeptide may undergo enzymatic processing , including removal of one or more amino acids, proteolytic cleavage and attachment of oligosaccharides

1. 2. 3. 4. 5. 6. 7.

Amino terminal and carboxy terminal modification Loss of signal sequences Attachment of carbohydrate side chain Attachment of isoprenyl group Addition of prosthetic group Proteolytic processing Formation of disulphide cross links

1.

2.

3.

Amino terminal and carboxy terminal modification: in fmet amino terminal residues may be removed enzymatically in formation of final functional protein. Carboxy terminal residues are also sometimes modified Loss of signal sequences: 15 to 30 residues at amino terminal end of some proteins play a role in directing the protein to its ultimate destination in the cell. Such signal sequences are removed by specific peptidases attachment of carbohydrate side chain: to glycoproteins enzymatically. These function extracellulary as well as lubricating proteoglycans that coat mucous membranes

4 addition of prosthetic groups: heme group of haemoglobin, biotin molecule of acetyl coA carboxylase 5 Proteolytic pocessing: many proteins are initially synthesised as large, inactive precursor polypeptides that are trimmed to form their smaller active forms e.g. proinsulin, chmyotrypsinogen 6 Formation of disulphide crosslinks:after folding into their native form , intra or inter chain disulphide bridges are formed between cys residues. These help to protect the native conformation of protein molecule from denaturation in extracellular environment.

Addition of isoprenyl group: a thioester bond is formed between the isoprenyl group and cys residue of the protein. Isoprenyl groups are derived from the pyrophosphorylated intermediates of the biosynthetic pathway and help to anchor the protein in a membrane. As a result transforming activity of ras oncogene is lost, when isoprenylation of ras protein is blocked use in cancer chemotherapy.

Thank you