Prokaryotic translation

It is the process of synthesis of protein by encoding information on mRNA.
Protein synthesis requires mRNA, tRNA, aminoacids, ribosome and enzyme aminoacyl tRNA synthase

1. PROKARYOTIC Translation Prasanna R Kovath Assistant Professor Department Of Biotechnology

2. T ranslation  is the process in which  ribosomes  in the  cytoplasm  or  endoplasmic reticulum  synthesize proteins after the process of  transcription  of  DNA  to  RNA  in the cell's  nucleus . The entire process is called  gene expression . Gene expression  is the process by which information from a  gene  is used in the synthesis of a functional  gene product  that enables it to produce end products, protein or  non-coding RNA ,

3. Translation : Overview Ribosomes bind to mRNA in the cytoplasm and move along the molecule in a 5’ – 3’ direction until it reaches a start codon (AUG) Anticodons on tRNA molecules align opposite appropriate codons according to complementary base pairing (e.g. AUG = UAC) Each tRNA molecule carries a specific amino acid (according to the genetic code) Ribosomes catalyse the formation of peptide bonds between adjacent amino acids (via condensation reactions) The ribosome moves along the mRNA molecule synthesising a polypeptide (protein) chain until it reaches a stop codon At this point translation ceases and the polypeptide (protein) chain is released The polypeptide chain synthesized with an amino end (N- terminal) and a Carboxyl (C-terminal) end.

4. Translation proceeds in three phases: Initiation : The ribosome assembles around the target mRNA. The first tRNA is attached at the  start codon . Elongation : The last tRNA validated by the  small ribosomal subunit  ( accommodation ) transfers the amino acid it carries to the  large ribosomal subunit  which binds it to the one of the precedingly admitted tRNA ( transpeptidation ). The ribosome then moves to the next mRNA codon to continue the process ( translocation ), creating an amino acid chain. Termination : When a stop codon is reached, the ribosome releases the polypeptide. The ribosomal complex remains intact and moves on to the next mRNA to be translated.

5. The key components of translation are: M essenger RNA   (goes to…) R ibosome   (reads sequence in …) C odons   ( recognised by …) A nticodons   (found on …) T ransfer RNA   (which carries …) A mino acids   (which join via …) P eptide bonds   (to form …) P olypeptides Mr Cat App

6. Amino acids : (20 in number forming the pool in the cytoplasm) Ribosome : ( exist as separate sub units,2 tRNA binding site: P site (peptidyl site) and A site (Aminoacyl site) E site also identified . Enzymes : Amino acyl tRNA synthetase, ( Amino acid activating system) Peptide polymerase (peptide bond formation between the amino acids bonded by their corresponding tRNAs to the ribosomal P site and A site, thus allowing sequential transfer of amino acids to the growing chain) Energy source : ATP, GTP for synthesis of peptide bond Soluble protein : for proper initiation, elongation and termination Inorganic Cations : (K+,NH+,NH4+,Mg2+)

7. Location I n  prokaryotes  (bacteria and archaea), translation occurs in the cytosol, where the large and small subunits of the  ribosome  bind to the mRNA. In  eukaryotes , translation occurs in the  cytoplasm  or across the membrane of the  endoplasmic reticulum  in a process called  co-translational translocation . In co-translational translocation, the entire ribosome/mRNA complex binds to the outer membrane of the  rough endoplasmic reticulum  (ER) and the new protein is synthesized and released into the ER; the newly created polypeptide can be stored inside the ER for future  vesicle  transport and  secretion  outside the cell, or immediately secreted.

8. Ribosomes Ribosomes are ribonucleoprotein particles that contain r-RNA and proteins. Each ribosome is made of two subunits. In prokaryotes, mitochondria and chloroplast of prokaryotes there is 70S ribosome which is composed of 50s and 30s subunits. In eukaryotes there is 80S ribosome which consists of 60s and 40s ribosomal subunit. 60s subunit consists of 28s rRNA (4718 nucleotides), the small 5s rRNA (120 nucleotides), 5.8s rRNA (160 nucleotides) and approximately 50 proteins. The 40s subunit consists of the 18s rRNA (1874 nucleotides) and 33 r-proteins. The 70s ribosome has three tRNA binding sites- P-site (or peptidyl-tRNA binding site), A-site (aminoacyl-tRNA-binding site), and E-site (deacylated tRNA, also called the exit site )

9. Structure of 70S Ribosome Structure of tRNA Structure of mRNA

10. Messenger RNA (mRNA) mRNA has a 5’ end, 5’ UTR, ribosomal binding site, coding sequence, 3’ UTR. In eukaryotes there are additional structures as 5’ Guanine cap and poly (A) tail . Messenger RNA (mRNA) has 3 reading frames out of which only one codes for desired protein. If in the sequence of bases there is no stop codon to interrupt the translation then that synthesis entire polypeptide chain and is that is called as open reading frames (ORF) .

11. Transfer RNA (t-RNA) Transfer RNA ( tRNA) has clover leaf structure in two dimension and L- shaped structure in 3 dimension. tRNA is 73 to 94 ribo -nucleotides in length. A tRNA molecule consists of 5’ phosphate terminal, an acceptor arm that ends in CCA terminal at 3’, D loop which often contains dihydrouridine, anticodon loop , and T arm which has TΨC where Ψ is pseudouridine . CCA sequence is important as it is important for recognition of tRNA and is also site of attachment of amino acid.

12. Each t-RNA is specific to amino acid that it carries it in CCA arm. There are 30-45 different tRNA in prokaryotes and 50 types in eukaryotes which suggest that there is more than one tRNA for single amino acid. Activation of amino acid: During this process amino acids are attached to the t-RNA in the presence of enzyme Amino acyl-t-RNA synthetase, this enzyme activate the amino acids by attaching covalently to the t RNA, when t RNA get charged, its named as aminoacyl-t RNA. During this process amino acids are attached to t-RNA with high energy bond, so called as activated amino acids. Amino Acids+ tRNA +ATP amino acyl t-RNA synthase Amino acyl-tRNA+AMP+PPi

13. Initiation in Prokaryotes Initiating amino acid Protein synthesis process involves step wise addition of aminoacids to the carboxy terminal end of the growing polypeptide chain that begins with an initiating aminoacid residue. The initiating aminoacid is a modified methionine residue encoded by initiation codon – (5’)AUG. Even though there is only one codon for methionine all organisms have 2 different t RNA for methionine amino acid- one for the initiating methionine residue (called tRNAfMet ) and the other for the internal methionine residues ( tRNAMet ). The N formyl methionine is incorporated in response to the initiating (5’)AUG codon while a methionine is incorporated in response to all other internal (5’)AUG codons.

14. Formation of N formyl methionine t RNA fMet ( fMet-tRNAfMet ) occurs in 2 steps. In the first step the amino acid methionine is attached to tRNAfMet in a reaction catalyzed by Met-tRNA synthetase. In E.coli the same enzyme is involved in aminoacylation of both tRNAfMet and tRNAmet . Methionine + tRNAfMet + ATP Met- tRNAfMet + AMP + PPi

15. In the second step a transformylase enzyme formylates the amino group of the methionine residue using N10-formyltetrahydrofolate as a formyl group donor. The enzyme transformylase in selective in formylating methionine attached to tRNAfMet , possibly by recognition its unique structural feature. This modification also allows fMet-tRNAfMet to bind to 30 S ribosomal P site which does not accept Met- tRNAMet or any other aminoacyl-tRNA. N10-Formyltetrahydrofolate + Met- tRNAfMet tetrahydrofolate + fMet-tRNAfMet

16. Initiation : Requirements 30S ribosomal subunit coding mRNA sequence Initiating fMet-tRNAfMet Three initiation factors (IF-1, IF-2, and IF-3) GTP (energy source) the 50S ribosomal subunit Mg2+ .

17. During the first step of initiation process the 30 S ribosomal subunit binds to the two initiation factors IF1 and IF3. IF3 prevents premature association of 30S and 50 S ribosomal subunits. The m RNA then binds precisely to the 30S ribosomal subunit such that the 5’AUG codon is placed at its P site . The intitiating AUG is directed to the appropriate location because of its close proximity to ShineDalgarno sequence. This consensus sequence consists of 4 to 9 purine bases, placed approximately 8 to 13 bp before of the 5’end of the initiation codon. The 16S rRNA of the 30S ribosomal subunit contains a pyrimidine rich series of bases near its 3’ end which is complementary and binds specifically to the Shine Dalgarno sequence allowing correct positioning of initiating 5’ AUG codon. The Shine Dalgarno sequence forms complementary base pairing with the pyrimidine rich series of bases towards the 3’end of the 16S ribosomal RNA of the 30S ribosomal subunit.

18. The bacterial ribosomes contain three binding sites for aminoacyl-tRNA, the the A site P site and E site that binds new incoming aminoacyl tRNA ( expection - the initiating aminoacyl tRNA binds to Psite ), peptidyl tRNA and deacylated tRNA respectively. The initiating 5’AUG positions itself correctly to the ribosomal P site which binds with the fMet tRNAfMet . The subsequent incoming aminoacyl-tRNA of the translation process binds first to the A site. The E site or the exit site is the site from where the uncharged tRNA leaves following peptide bond formation during elongation. In the next step of initiation a complex consisting of GTP bound IF2 and fMet-tRNAfMet associate with the complex of ribosomal 30S subunit, IF1, IF3 and mRNA. The anticodon of the tRNA pairs correctly with the initiating 5’AUG positions at the ribosomal P site.

19. In the third step a the ribosomal 50S subunit associates with the complex which is accompanied with the GTP hydrolysis into GDP and Pi along with the release of the 3 initiating factors . This results in a functional 70S initiation complex associated with the mRNA and the initiating f Met tRNA fMet . This is now ready to continue with the elongation steps .

20. 3 different interaction ensure proper binding of intiating fMet -tRNA f Met to ribosomal P site Codon anticodon interaction between the fMet tRNA fMet and intiating 5’AUG codon. 2) Interaction between the Shine –Dalgarno sequence and the 16SrRNA ribosomal sequence. 3) Binding interactions between the fMet tRNA fMet and the ribosomal P-site.

21. https://youtu.be/KZBljAM6B1s

22. The  Shine–Dalgarno  ( SD )  sequence  is a  ribosomal binding site  in bacterial and archaeal  messenger RNA , generally located around 8 bases  upstream  of the  start codon  AUG.  The RNA sequence helps recruit the  ribosome  to the  messenger RNA  (mRNA) to initiate  protein synthesis  by aligning the ribosome with the start codon. Once recruited,  tRNA  may add amino acids in sequence as dictated by the codons, moving downstream from the translational start site. The Shine–Dalgarno sequence is common in  bacteria , but rarer in  archaea .  It is also present in some  chloroplast  and  mitochondrial  transcripts. The six-base  consensus sequence  is  AGGAGG ; The Shine–Dalgarno sequence was proposed by  Australian scientists  John Shine  and  Lynn Dalgarno .

23. Elongation : Requirements Initiation complex A minoacyl-tRNAs, E longation factors: (EF-Tu, EF-Ts, and EF-G in bacteria) EFTu : Directs the next tRNA to its correct position in the ribosome. EF-Ts : Regenerates EF-Tu after the latter yielded the energy contained in its attached GTP molecule. EF-G : Mediates translocation. E nergy from GTP hydrolysis Elongation B inding of the second aminoacyl-tRNA/Positioning a second transfer RNA P eptide bond formation Translocation

24. 1.First elongation step : binding of the second aminoacyl-tRNA/Positioning a second transfer RNA The subsequent aminoacyl-tRNA binds to the A site of ribosomes. The appropriate aminoacyl-tRNA associates with a GTP-EF-Tu complex resulting in formation of aminoacyltRNA –EF-Tu–GTP complex. It binds to the ribosomal A site with simultaneous hydrolyzed of GTP and an EF-Tu–GDP complex is released from the 70S ribosome. The EF-Tu–GTP complex is then reformed in a reaction involving EF-Ts and GTP.

25. 2.Peptide bond formation P eptide bond formation takes place among the amino acids attached to the ribosomal P and A site by their respective t- RNA molecules. During this step the amino group of aminoacid at the ribosomal A site makes a nucleophilic attack and displaces the tRNA in the Psite resulting in peptide bond formation between the 2 aminoacids . The 1 st Amino acid (N- formylmethionine ) is removed from its attachment to its tRNA and transferred to the free- NH2 terminus of second aminoacid.The first aminoacid is thus placed “ on top of ’’ This results in formation of dipeptidyl t-RNA at the A site of ribosomes and a deacylated tRNA at the P site of ribosomes. The reaction is catalyzed by enzyme peptidyl transferase which is now known to be catalyzed by the ribosomal 23srRNA subunit of ribosome.

26. binding of the second aminoacyl-tRNA. formation of the first peptide bond

27. 3.Translocation Elongation Factor EF- G,earlier called translocase,catalyses the translocation process. The ribosome must be converted from pretranslocational state to the post translocational state by the action of EF-G T he ribosome moves by a distance of one codon towards the 3’ direction of the mRNA, So that the codon enters the A site. T he dipeptidyl tRNA in A site moves to the P site of ribosomes T he deacylated tRNA in the P Site moves to the E site of ribosomes, from where the tRNA is released into the cytosol. For each amino acid molecule added to the growing polypeptide chain 2 GTP molecules are hydrolyzed into GDP and Pi.

28. Translation Termination

29. Termination The Elongation process continues until the ribosomes encounters a termination codon on the m RNA molecule. There are three codons that signal translation termination, these are UAG, UAA, and UGA. Presence of one of these codons after the final coded amino acid acts as a translation termination signal. Requirements T ermination factors, or release factors RF-1 : Recognizes termination codons UAA and UAG RF-2 : Recognizes termination codons UAA and UAG RF-3 : Stimulates dissociation of RF 1 and RF 2 from the ribosome after termination

30. In bacteria, when the termination codon are positioned at the A site of the ribosomes the termination factors, or release factors ( RF-1, RF-2, and RF-3) carry out breakdown of the bond between polypeptide and the tRNA molecule, releasing free polypeptide and tRNA molecule. The 70S ribosomal subunit then dissociate into 30S and 50S subunits to begin with a new protein synthesis cycle. Termination codons UAG and UAA, are recognized by RF-1 and UGA and UAA are recognized by RF-2. Depending on the codon present appropriate release factor binds it, and brings about hydrolysis of bond between the polypeptide and tRNA molecule.

31. Modifications of Amino-Terminal and Carboxyl-Terminal amino acids In bacteria the first amino acid in all polypeptide chains is a N- formylmethionine residue. The formyl group and the initiating methionine and sometimes further amino terminal and carboxy terminal aminoacids are enzymatically removed from the final protein structure. Loss of Signal Sequences Signal sequences range from 15 -30 residues in length and play an crucial role in targeting of the protein to their location in the cell. Once the protein reaches its final location the signal sequences are enzymatically cleaved and are not the part of the final functional protein. Post Translational modifications

32. Modification of individual aminoacids Bacteria modify several amino acid side chains of proteins by addition and removal of phosphate groups by enzymes names kinases and phosphatases respectively. In bacteria kinases often phosphorylate histidine and aspartate residues which are important modification in bacteria two component regulatory systems. A classic bacteria two component system comprises of a sensor protein which contains a histidine kinase domain that autophosphorylates the histidine aminoacid in response to a signal. The kinase then transfers the acquired phosphate residue to the aspartate residue of the second component which is known as the response regulator. The phosphorylated regulator then controls the transcription of several downstream genes thus operating a regulatory cascade .

33. Protein glycosylation in bacteria: It is a commonly post translational modification in bacteria. Many surface appendage proteins like pilin of pili and flagellin of flagella contains glycosylated residues. The process has been described in both Gram positive and Gram negative bacteria and may play an important role in adhesion, stabilization of proteins against proteolysis and evasion of host immune system. The commonly glycosylated amino acid residues includes serine and threonine residues which are O glycosylated and asparagine residue which is N glycosylayted .

34. Addition of Prosthetic Groups Prosthetic group is a non-protein component of some proteins that is required for the their activity. Prosthetic group may be organic or inorganic in nature but are never made up of amino acids. These are tightly bound to the protein component through covalently bound. For example – Ferrodoxins are a family of bacterial proteins containing 2, 4 or 8 atoms of iron and additional inorganic sulphate. In bacteria ferrodoxins are components of electron transport chain during processes such as nitrate, nitrite and sulphate reduction.

35. Proteolytic Processing Many proteins are produced as large inactive forms of proteins known as precursor. Later during the post translational modifications these are proteolytically cleaved into smaller active form of the protein. Many bacterial toxins achieve their high potency by delivery of the catalytically active polypeptide fragment of the toxin to the eukaryotic cell cytosol. Activation occurs by proteolytic cleavage of the polypeptide at the defined site. Examples include Diptheria toxin, anthrax toxin, etc.

36. Formation of Disulfide Cross-Links Post translationally many protein fold into its native conformation many proteins from intrachain or interchain disulfide bonds between their cysteine residues. These bonds are sometimes important in formation of the final functional active protein molecule. Alkaline phosphatase enzyme from E.coli is a homodimer where in two intramolecular disulfide bonds are involved in formation dimeric protein with full enzymatic activity.