Biosynthesis of a protein or a polypeptide in a living cell -translationThe synthesis of every protein molecule in a cell is directed by an m RNA intermediate which is copied from DNA by
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Important points about translation
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Biosynthesis of a protein or a polypeptide in a living cell -translationThe synthesis of every protein molecule in a cell is directed by an m RNA intermediate which is copied from DNA by transcription
?Wide variation in protein synthesis in cells depending on the need and ability of cells?Erythrocytes -lack translation machinery and cannot synthesis proteins?Growing and dividing cells produce large quantities of proteins
?Proteins are high molecular weight ?N containing organic compounds of complex shape and composition.?It consists of one or more macro molecular subunits called polypeptides,which are composed of smaller building blocks amino acids.?Amino acids of a polypeptide are joined by a peptide bond.?Peptide bond is a covalent bond formed between the carboxyl group of one amino acid and amino group of an adjacent amino acid
??Triplet (3 nucleotide) base sequences in mRNA that act as code words for amino acids in protein constitute the genetic code??Codons composed of 4 nucleotide bases A,G (purines) and C ,U (pyrimidines)??4 bases produce 64 combinations (43)??Codons on mRNA is written from 5 to 3 end??61 codons code 20 aminoacidsin protein??3 codons UAA, UAG & UGA stop codons -do not code for amino acids. Also called termination codons or non-sense codons??UAG,UAA & UGA amber, ochre & opal resp.??AUGchain initiating codon or start codon (GUG in some cases)
1.The codon is a triplet code:Each codon that specifies an amino acid in a polypeptide chain consist of 3 nucleotides.2.The codon is continous:The codon is read continuosly-3 nucleotides at a time without skipping any nucleotides3.The codon is non overlapping :The codon is read in successive groups of 3 nucleotides without any comma, punctuationsEg: UUUCUUAGAGGG is read as UUU /CUU/AGA /GGGAddition or deletion of 1 or 2 bases will change the sequence in mRNA and the protein synthesisedwill be totally different (frame shift mutation)
4.The code is almost universal :??Same codon codes for same aminoacidsin all living organisms. Thus the genetic code has been conserved during evolution??Exception : AUA codes for methioninein mitochondria, the same codon (AUA) codes for isoleucinein cytoplasm5. The codon is specific :??A particular codon always codes for the same aminoacid. Hence genetic code is highly specific and unambiguous . Eg: UGG codes for tryptophan
5.The codon is degenerate :With 2 exceptions (only AUG codes for methionineand only UGG codes for tryptophan) more than one codon occurs for each amino acid.This multiple coding is called the degeneracyof the code.Glycinehas four codons. The codons that designate the same amino acid are called synonyms. Most synonyms differ in the 3rdbase of the codonEg; when first 2 nucleotides in a codon are identical and 3rdletter is U or C ,the codon always codes for same amino acidi.e., UUU,UUC -phenyl alanineCAU,CAC -histidine
6.The code has start and stop signals :Specific start and stop signals for protein synthesis are contained in the code.In both eukaryotes and prokaryotes AUG (methionine)is almost always the start codon for protein synthesis.Only 61 of the 64 codons specify amino acids; these codons are called sense codons.The other 3 codons UAG, UAA, UGA do not specify an amino acid.Theseare stop codons or nonsense codonsor terminating codons.
?Put forth by Crick?The codon of mRNA is recognized by the anticodon of tRNAmRNA 5 to 3tRNA 3 to 5 ?First 2 bases of codon base pair with last 2 bases of anticodon by usual conventional complementary base pairing?3rdbase of codon is flexible with regard to complementary base
Wobble hypothesis is the phenomenon in which a single tRNA recognize more than one codon. This is due to the fact that the 3rdbase in the codon (3) often fails to recognize the specific complementary base in the anticodon (5)anticodon codonC GA U conventional base pairingU G or AG Uor C non conventional ( colored)
One t RNA can pair with multiple m RNA codon because of wobble or less stringent or flexible binding of the third nucleotide base of the codon to the first base of the t RNA anticodon.The complete set of 61 sense codons can be read by fewer than 61 distinct t RNAs because of pairing properties of the bases in anticodon
Protein synthesis involves the translation of nucleotide base sequence of mRNA into the language of amino acid sequenceCOMPONENTS OF TRANSLATION : * Amino acids* Ribosomes* mRNA ( messenger RNA )* tRNA ( Transfer RNA )* Energy sources* protein factors
1.AMINO ACIDS :?Proteins are polymers of amino acids?20 amino acids?10 essential amino acids supplied through diet?Protein synthesis occur only when all the amino acids needed for a particular protein synthesis are available?Deficiency of any one essential amino acid translation stops
2. RIBOSOMES :?centre for protein synthesis?Prokaryotes 70S & Eukaryotes 80S?Has 2 subunits one big & one small?Each ribosome has a. Peptidyl site (P site) : where t RNA carrying the growing polypeptide chain resides ( binding of peptidyl tRNA ). Amino acylsite (A site): binding of amino acyltRNA . Exit site (E site): from which t RNAs leave the ribosome after they have discharged their amino acids.?Prokaryotes -A & P sites?Eukaryotes -A , P & E sites
The prokaryotic ribosome is an extraordinarily complex organelle made of a 30S and a 50S subunitEach subunit is constructed from one or two rRNAmolecules and many polypeptides.Eukaryotic cytoplasmic ribosomesare 80S 40S & 60 SStructure of prokaryotic ribosome
P siteA siteE sitePolyribosomeseveral Ribosomes simultaneously translate on a single mRNA
3. Messenger RNA (mRNA) :?Information for protein synthesis is present on mRNA?DNA pass genetic information to mRNA as codons ?Prokaryotic mRNA polycistronica single mRNA has many coding regions that code for different polypeptides?Eukaryotic mRNA monocistroniccodes for a single polypeptide
4. Transfer RNA (tRNA):?Carry the amino acids and hand them over to the growing peptide chain?The amino acid is covalently bound to tRNA at the 3 end?Each tRNA has an anticodon which recognize the codon of mRNA for protein synthesis?Man 50 tRNAs?Bacteria 40 tRNAs
5. ENERGY SOURCES :?ATP & GTPSupplies energy for translation?Both breakdown to AMP & GMP resp with the liberation of pyrophosphate6. PROTEIN FACTORS :?Requires many protein factors at different stages of translation?Includes Initiation,Elongation&Release factors?These molecules are proteins needed at particular stages of polypeptide synthesis
??Activation & attachment of amino acids to tRNAs2 step process enzyme involved is amino acyltRNA synthetase??Amino acid first attached to enzyme utilizing ATP to form enzyme-AMP-aminoacid complex??The amino acid is then transferred from the complex to the 3 end of tRNA to form amino acyltRNA. The enzyme and AMP will be released
?The translation process can be subdivided into 3 stages .1.Initiation2.Elongation3.Termination?The mRNAis read in the 5 to 3 direction & the polypeptide synthesis proceeds from N-terminal end to C-terminal end
Initiation stage : m RNA is bound to the ribosome and positioned for proper translationElongation stage: amino acids are sequentially joined together via peptide bonds in an order ,specified by the arrangement of codons in m RNA.Termination stage: m RNA and the newly formed polypeptide chain are released from the ribosome.
INITIATION??Requires initiation factors , ribosomal subunits , m RNA and initiator RNA.PROKARYOTIC INITIATION??The initiation can be sub divided in to 3 distinct steps.Step 1:?3 initiation factors called IF1,IF2,and IF3bind to the small(30 s ) subunit ,with GTPattaching to IF2.Step 2:?mRNA & initiator tRNAbind to the 30 s subunit of ribosome by means of a special nucleotide sequence called the mRNAs ribosome binding site(Shine Dalgarnosequence).This sequence consists of a stretch of 3-9 purinenucleotides(often AGGA) located slightly upstream of the initiation codon.
Shine-Dalgarnosequence
InitiatortRNA-tRNAfMet-carriesN-formylmethionine(fMet)InN-formylMet,theaminogroupofmethionineisblockedbytheadditionofaformylgroupandonlythecarboxylgroupisavailableforbondingtoanotheraminoacid.HenceN-formylMetcanbesituatedonlyattheNterminalendofapolypeptidechain.
During initiation ,the initiator tRNA is bound to the P-site of the 30s subunit by the action of IF2(bound to GTP)Once t RNA fMetenters the P site ,its anticodon base pairs with AUG start codon in the mRNA and IF1 and IF3 are released.Thus the 30 s subunit, IF2-GTP,m RNA and t RNA fMetis called the 30s initiation complex
Step 3 The 30S initiation complex is joined by a free 50S ribosomal subunit , generating the 70 S initiation complex.Binding of the 50S subunit is driven by hydrolysis of the GTP associated with IF2 and IF2 leaves the ribosome. At this stage all initiation factors are released
Elongation cycle composed of three phases: aminoacyl tRNA binding, the transpeptidation reaction, and translocationThe process is aided by special protein elongation factorsAt the beginning of an elongation cycle, the peptidyl (P) site is filled with either t RNA fMetor peptidyl-tRNA and the aminoacyl (A site) and exit site (E site) are empty
1. Aminoacyl-tRNA binding phase:?First phase of elongation cycle ?An aminoacyltRNAbinds to the A site, escorted by EF-Tubound to GTP ?During tRNAbinding GTP is hydrolyzed and EF-Tuis released ?EF-Ts helps recycle the EF-Tu
2. Transpeptidation reaction : ?By the enzyme -peptidyltransferase, located on the 50S subunit?Catalyses peptide bond formation between the amino group of amino acid in the A site and the -carboxyl group of the amino acid on the P site?No extra energy source is required?The net result of peptide bond formation is the attachment of the growing peptide chain to the tRNA in the A-site
3. Translocation : ??The growing peptide chain (peptidyl-tRNA) moves from the A site to the P site and the empty t RNA moves from the P site to the E site, where it is released from the ribosome.??the ribosome moves one codon along mRNA so that a new codon is positioned in the A site??This process requires an elongation factor called EF-Gor translocaseprotein,together with a bound molecule of GTP, become transiently associated with the ribosome.??The net effect of translocation is to repeat the elongation cycle??The ribosome changes shape as it moves down the mRNA in the 5? to 3? direction
Protein synthesis stops when the ribosome reaches one of three special nonsense codons?UAA, UAG, and UGAThree release factors (RF-1, RF-2, and RF-3)aid the ribosome in recognizing these codons.Peptidyl transferase hydrolyzes the peptide to free from its tRNA, and the empty tRNA is released.TheribosomedissociatesfromitsmRNAandseparatesinto30Sand50Ssubunits.IF-3bindstothe30Ssubunitandpreventsitfromre-associatingwiththe50Ssubunituntiltheproperstageininitiationisreached.GTPhydrolysisisprobablyinvolved
?Folding of newly synthesized polypeptides chaperones?Not bind to normal proteins?Recognize only unfolded proteins or partly denatured proteins?Suppress incorrect folding & may reverse any incorrect folding ?Present in all cells prokaryotes & eukaryotesIn E coli 4 chaperones -DnaK, DnaJ, GroEL, and GroES?and the stress protein GrpE.Also called heat shock proteins or stress proteinsthey increase in concentration when cells were exposed to high temp., metabolic poisons & other stressful conditions
1. Hsp70 system : . Consists of Hsp70 & Hsp40. Can bind individually to the protein and help in the correct formation of protein folding2. Chaperonin system:. Large oligomericassembly which forms a structure into which the folded proteins are inserted. Hsp60 & Hsp10
??Polypeptide chains emerged from ribosome is inactive& before taking on its functional role in the cell must undergo modifications known as post translational modificationsor processing (PTM)??Includes . Protein folding. Trimming by proteolytic degradation. Inteinsplicing. Covalent modifications
Proteolytic degradation:?proteins synthesized as precursors zymogensbigger than the functional proteins?Some portions of this precursors are removed by proteolytic cleavage to liberate active proteins -trimming?Carried out by proteases or peptidases?Eg: 1. formation of insulin from preproinsulin2. conversion of trypsinogento trypsin
Insulin-produced by islets of langerhansin inactive pre-pro insulin form(110aa)Pre-pro to pro insulin (86aa) removing leader or signal peptide (N-terminal, first 24 aminoacids)Pro insulin to active form (51aa) removing connectingsequence& linking A & B chains by 2 disulphide bonds
Inteinsplicing:??Inteinsintervening sequences (intronsin mRNA)??Inteinsremoved and exteinsligatedin appropriate order for the protein to become active
Covalent modifications :??Proteins synthesized in translation under go covalent changes modifications in amino acids to become active or inactivea) Phosphorylation.OH group containing aminoacidsof proteins ser, threonineand tyrundergo phosphorylation. Phosphorylation may increase or decrease protein activityprotein kinases -phosporylationprotein phosphatases-dephosphorylation
b) Hydroxylation. In collagen formation proline& lysine converted to hydroxyproline& hydroxylysine resp.. Occurs in endoplasmic reticulum and requires vitamin Cc) Glycosylation. Attachment of carbohydrate moiety to proteins. Mainly attached to serine and threonine(O-linked) or to asparagine(N-linked) . Leads to glycoproteinssynthesis
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