Translation in prokaryotes

this slide provide a basic view on translational mechanisms in prokaryotes

1. Translation in Prokaryotes Submitted to – Dr. Umesh Gautam Submitted by- Jaskirat Singh ( 11715744) Navdeep Singh (11700891) Thounaojam Ronald (11715437) Smita Bhattacharjee (11713639) Section – B1724

2. Translation The genetic information contained within the order of nucleotides in the messenger RNA (mRNA) is used to generate the linear sequences of amino acids in proteins. These linear chains of amino acids are further processed and modified.

3. Translation is highly conserved among all the organisms and is energetically costly for the cells. The machinery responsible for translating the lanaguage of messenger RNA into the language of proteins It occur in cytoplasm where ribosomes are located It is a universal process

4. Machinery of Translation mRNA tRNA Aminoacyl tRNA synthetases Ribosome The proteins coding region of mRNA consists of an ordered series of three nucleotide long units called Codons .

5. Steps of translation Activation of amino acids Charging of tRNA (loading of tRNA ) TRANSLATION Initiation of polypeptide chain Elongation of polypeptide chain Termination of polypeptide chain

6. Activation of amino acids 20 types of amino acids partcipate in protein synthesis Amino acids reacts with ATP to form “ amio acyl AMP enzyme complex” which is also known as “ activated Amino acid” This reaction is catalyzed by specific ‘ amino acyl tRNA synthetse ’ enzyme

7. Speific amino acyl tRNA synthetse ’ enzyme for each amino acid

8. Charging of t-RNA Specific amino acid is recognized by its specific tRNA Now amino acid attaches to the ‘ amino acid attachement site’ of its specific t-RNA and AMP and Enzyme are separated from it. Amino acyl t-RNA complex is also called ‘ charged t-RNA’

9. Now amino acyl t-RNA moves to the ribosome for protein synthesis,

10. Translation - 3 steps Initiation of polypeptide chain Elongation of polypeptide chain Termination of polypeptide chain

11. Initiation of polypeptide chain In this step 30’s’ and 50’s’ sub units of ribosome, GTP, Mg2+, charged t-RNA, mRNA and some initiation factors are required In prokaryotes there are three initiation factors IF 1, IF2, IF3 Initiation factors are specific protiens GTP and initiation factors promote the initiation process.

12. Both subunits of ribosomes are separated with the help of IF3 factor In prokaryotes with the help of “S D sequence mRNA recognises the smaller subunit of the ribosome. A sequence of 8 N2 base is present before the 4-12 N2 base of initiation codon og mRNA called SD sequence. In smaller subunit of ribosome, a comlementary sequence of SD sequence is present on 16’s’ rRNA , which is called as anti shine delgarno sequence.

13. With the help of SD and ASD sequence of rRNA mRNA recognises smaller unit of ribosomes This 30’s’ mRNA complex reacts with ‘ formyl methionyl tRNA complex’ and “ 30’s’ mRNA fromyl metthionyl tRNA complex” is fromed . This tRNA attaches with codon parrt of mRNA . A GTP molecule is required.

14. Now the larger subunit of ribosome (50s sub unit) joins the complex. The initiation factor released and complete 70s ribosome is formed. In larger sub unit of ribosome there are three sites for tRNA ‘ P site’ = peptidtyl site ‘ A site’ = amino acyl site ‘ E site ’ = Exit site

15. ELONGATION ( prokaryotes )

16. Elongation – It’s the addition of amino acids by the formation of peptide bonds . Elongation is a chain of amino acid eventually forming a polypeptide bond . Steps of elongation – Elongation mainly consists of two steps :- Binding of new Aminoacyl t-RNA : codon in m-RNA determines the incoming AA. EF 1 & GTP required. PEPTIDE BOND FORMATION : alpha- NH2 group of incoming AA in A site forms peptide bond with COOH group of AA in P site . Enzyme is Peptidyl Transferase . No need for energy as AA is activated.

17. Diagram

18. Step 1 The second charged tRNA binds to the first ribosome at the latter’s ‘A’ site with the help of the proteins ,called elongation factors (EF- Tu ). EF- Tu carries a molecule of GTP. Correct hydrogen bonding with the mRNA template dictates the selection of a new tRNA , and the activity of ghe EF- Tu ensures the proper positions of the tRNA in the A site. After performing its function , the EF- Tu protein dissociates from the ribosome , and in the cytoplasm is subsequently regenerated to its active form by another elongation fator , the EF-Ts. At this point , both sites of the ribosome are occupied by the tRNA’s ,each of which carries an amino acid ,and each of which is hydrogen bonded to the template mRNA.

19. Ist step

20. 2 nd step The next step is the formation of a peptide bond between the two amino acids. To accomplish this job , the first amino acid ( N- formylmethionine ) is removed from its attachment to its tRNA and transferred to the free-NH2 terminus of the second amino acid . The first amino acid is thus placed on the top of second amino acid. The resulting compound is a dipeptide whose carboxyl end is still bonded to the second tRNA , but whose amino end ids free. The reaction is catalysed by an enzyme associated with the 50S subunit and called peptidyl transferase . The energy for peptide bond formation is supplied by the dissolution of the aminoacyl bond between the first amino acid and its carrier tRNA,this energy having originally been donated by ATP .

21. 3 rd step (translocation) Although a dipeptide has been generated ,continued synthesis requires that the next codon be made available and that the next tRNA be admitted to the A site on the ribosome : this site being still occupied by dipeptide-carrying tRNA . The problem is solved by a movement of the entire ribosome relative to the mRNA strand. T his is called translocation and consists of following three steps- Ejection of discharged tRNAfmet from the P site . Movement or physical shifting of tRNA dipeptide from the ‘A’ site to the P site. Movement of the mRNA is such that the effect is the apparent movement of the ribosome in 5’→3’ direction by the length of one codon (3 nucleotide) . This step requires the presence of an elongation factor EF-G called translocase and GTP.

22. Contd . The sequential formation of a polypeptide continues in the manner described above. A tRNA in the p site shifts its burden of growing polypeptide to the next succeeding tRNA,followed by translocation ,exit of the discharged tRNA ,and entrance of a new charged tRNA to base pair with anew codon at A aite . Thus, the growing polypeptide is adopted in turn by each tRNA ,with each successive amino acid being added in effect , to the bottom of stack . As process continues , the mRNA ia progressively translated from 5’→3’ end.

23. Termination

24. This is the last phase of translation. Termination occurs when one of the three termination codons moves into the A site.  These codons are not recognized by any tRNAs . Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons ) or RF2 (recognizing the UAA and UGA stop codons ). These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process.