protein synthesis in prokaryotes.pptx

presentation covers basic structure of protein, its synthesis and regulation in prokaryotic organisms

1. PROTEIN SYNTHESIS IN PROKARYOTES : Structure, synthesis and its reguation Dr.Vineetha P G 22-DVM-03, PhD Scholar Dept. Poultry Science CVAS, Mannuthy

2. Protein structure is defined as a polymer of amino acids joined by peptide bonds

3. This bond is otherwise an amide linkage . When peptide bonds are established among more than ten amino acids-form a polypeptide chain. When number of amino acids in the polypeptide chain exceeds 100 -forms a protein

4. Classification of Proteins Based on the molecular shape, proteins can be classified into two types. 1 . Fibrous Proteins : When the polypeptide chains run  parallel  and are held together by hydrogen and disulfide bonds, then the fiber-like structure is formed. These are water-insoluble proteins. Example   – keratin (present in hair, wool, and silk) and myosin (present in muscles), etc.

5. 2. Globular Proteins : This structure results when the chains of polypeptides  coil around  to give a spherical shape. These are usually soluble in water. Example   – Insulin and albumins are common examples of globular proteins.

6. Levels of Protein Structure Linderstrom -Lang (1952) -suggested a hierarchy of protein structure with four levels: primary, secondary, tertiary , and quaternary. Primary Structure of Protein The Primary structure of proteins is the exact ordering of amino acids forming their chains. Covalent, peptide bonds which connect the amino acids together maintain the primary structure of a protein.

7. Secondary Structure of Protein These polypeptide chains fold due to the interaction between the amine and carboxyl group of the peptide link. They are found to exist in two different types of structures  α – helix and β – pleated sheet structures . This structure arises due to the regular folding of the backbone of the polypeptide chain due to hydrogen bonding between -CO group and -NH groups of the peptide bond.

8. (a) α – Helix:  α – Helix -a polypeptide chain forms hydrogen bonds by twisting into a right-handed screw with the -NH group of each amino acid residue hydrogen-bonded to the -CO of the adjacent turn of the helix. (b) β – pleated sheet:  The polypeptide chains are stretched out beside one another and then bonded by intermolecular H-bonds . The structure resembles the pleated folds of drapery and therefore is known as β – pleated sheet

9. Tertiary Structure of Protein This structure arises from further folding of the secondary structure of the protein. H-bonds, electrostatic forces, disulphide linkages, and Vander Waals forces stabilize this structure. It gives rise to two major molecular shapes called fibrous and globular.

10. Quaternary Structure of Protein Some of the proteins are composed of two or more polypeptide chains referred to as sub-units. The spatial arrangement of these subunits with respect to each other is known as quaternary structure. A protein’s shape is determined by its primary structure (the amino acid sequence).

11. TRANSCRIPTION Transcription   is the process of copying DNA, the master instructions for the cell, into another molecule called mRNA. In prokaryotes, transcription occurs in the  cytoplasm RNA polymerase is the protein that actually does the copying of DNA. DNA is one long strand in prokaryotes, with coding genes. Each gene codes for one protein. In prokaryotes, genes are organized into groups called  operons . Eg:These genes code for proteins that are used for a common function, like metabolizing the sugar lactose, so they're all grouped together.

12. In prokaryotes, post-transcriptional changes aren’t needed- transcription produces mature mRNA instantly. Ribosomes create polypeptide chains from mRNA during translation. Transcription and translation occur in the cytoplasm of nucleus-less prokaryotes.

13. Proteins are the active players in most cell processes , implementing the myriad tasks that are directed by the information encoded in genomic DNA. Protein synthesis is thus the final stage of gene expression. The polypeptide chain fold into the three-dimensional conformation undergo various processing steps before being converted to its active form.

14. Operon François  Jacob  and Jacques  Monod   - showed the organization of bacterial genes into operons -studies on the  lac   operon  of  E. coli . In   E. coli , all structural genes encode enzymes needed to use lactose lie next to each other in the lactose (or  lac ) operon under the control of a single promoter, the  lac  promoter. They won the Nobel Prize in Physiology or Medicine in 1965.

15. In prokaryotes, operons whose gene products are required consistently – expression is unregulated- transcribed and translated to the protein products. Such genes encode enzymes in housekeeping functions -cellular maintenance, including DNA replication, repair, and expression , enzymes involved in core metabolism. Prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers

16. Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region. The regulatory region includes the promoter , to which  transcription factors can bind. Transcription factors influence the binding of  RNA polymerase  to the promoter - to transcribe structural genes.

17. Transcription The process of synthesis of RNA by copying the template strand of DNA is called transcription. During replication -entire genome is copied but in transcription - the selected portion of genome is copied. The enzyme involved is RNA polymerase.

18. THREE steps involved Initiation Closed complex formation Open complex formation Tertiary complex formation Elongation Termination

19. Initiation The transcription is initiated by RNA polymerase holoenzyme from a specific point called promotor sequence. Bactreial RNA polymerase consists of α, β, β’ and ω sub units. The binding of core polymerase to promotor is facilitated and specified by sigma (σ) factor. (σ70 in case of E. coli).

20. The core polymerase along with σ-factor is called Holo -enzyme ie . RNA polymerase   holoenzyme . In case of e. coli, promotor consists of two conserved sequences 5’-TTGACA-3’ at -35 element and 5’-TATAAT-3’ at -10 element. Binding of holoenzyme to two conserve sequence of promotor form close complex.

21. After formation of closed complex, the RNA polymerase holoenzyme separates 10-14 bases called melting. So that open complex is formed. This changing from closed complex to open complex is called  isomerization . RNA polymerase starts synthesizing nucleotide. It does not require the help of primase .

22. Elongation After synthesis of RNA more than 10 bp long, the σ-factor is ejected and the enzyme move along 5’-3’ direction continuously synthesizing RNA. The synthesized RNA exit from RNA exit channel. The synthesized RNA is proof reads by Hydrolytic editing. For this the polymerase back track by one or more nucleotide and cleave the RNA removing the error and synthesize the correct one. The Gre factor enhance this proof reading process. Pyrophospholytic editing another mechanism of removing altered nucleotide  

23. Termination  Rho independent: In this mechanism, transcription is terminated due to specific sequence in terminator DNA. The terminator DNA contains invert repeat which cause complimentary pairing as transcript RNA form hair pin structure. This invert repeat is followed by larger number of TTTTTTTT(~8 bp ) on template DNA. The uracil appear in RNA. The load of hair pin structure is not tolerated by A=U base pair so the RNA get separated from RNA-DNA heteroduplex .

24. Rho dependent In this mechanism, transcription is terminated by rho (ρ) protein. It is ring shaped single strand binding ATpase protein. The rho protein bind the single stranded RNA as it exit from polymerase enzyme complex  and hydrolyse the RNA from enzyme complex.

25. The three post-transcriptional modifications are as follows: Splicing :   removal of the non coding regions ( introns ) and joining all coding genes ( exons ) to form a functional gene. Capping :   addition of nucleotide methylguanosine triphosphate to the 5'-phosphate end of the mRNA. It guides the mRNA safely to the cytoplasm upon its exit from the nucleus. Tailing :  This involves the addition of poly-adenosine residues to the 3'-end of the mRNA .

26. Regulation of Gene Expression Replication level  – Any error in copying the DNA -an altered expression. Transcriptional level  –any error in the polymerization -change in expression of the gene. Post-transcriptional level  – During RNA splicing, there may be some changes. Translational level  –if there is an error in the attachment of mRNA to the tRNA molecules, there may arise some changes.

27. Regulation of transcription process Repressors  are proteins that suppress transcription of a gene in response to an external stimulus. In other words, a repressor keeps a gene “off.” Activators  are proteins that increase the transcription of a gene in response to an external stimulus. In other words, an activator turns a gene “on.” Inducers  are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate. Inducers basically help speed up or slow down “on” or “off” by binding to a repressor or activator. In other words: they don’t work alone.

28. Regulation by Repression Repression is the process whereby a repressor inhibits the transcription of an operon . The repressor is usually an amino acid, and the proteins produced from the repressible operon Working of a Repressor (1) The active repressor binds to the operator. (2) RNA polymerase, therefore, cannot bind to the promoter, and the operon is not transcribed. (3) The cell stops producing the structural proteins encoded by the operon . For Example: If an amino acid is present in the medium, E. coli does not need to synthesize that amino acid and cease to produce the enzymes required for its synthesis. The Tryptophan operon is repressible.

29. Regulation by Catabolite Repression Some operons (e.g., lac and ara ) are not expressed when glucose is present in the medium. These operons require cAMP for their expression. Working: (1) Glucose causes cAMP levels in the cells to decrease. (2) When glucose decreases, cAMP levels rise. (3) cAMP binds to the catabolite -activator protein (CAP). (4) The cAMP –protein complex binds to a site near the promoter of the operon and facilitates binding of RNA polymerase to the promoter. Example: The lac operon exhibits catabolite repression. In the presence of lactose and the absence of glucose, the lac repressor is inactivated , and the high levels of cAMP facilitate the binding of RNA polymerase to the promoter. The operon is transcribed, and the proteins that allow the cells to utilize lactose are produced.

30. Regulation by Induction Induction is the process whereby an inducer (a small molecule) stimulates the transcription of an operon . Working of an Inducer: (1) The inducer binds to the repressor, inactivating it. (2) The inactive repressor does not bind to the operator. (3) RNA polymerase, therefore, can bind to the promoter and transcribe the operon . (4) The structural proteins encoded by the operon are produced. Example: If glucose is not present in the provided medium but another sugar is available, E. coli produces the enzymes to utilize that sugar. The Lac operon is inducible.

31. TRANSLATION

32. Following transcription is translation-production of proteins based on mRNA blueprints. Components - Ribosomes , initiation factors, elongation factors, amino acids, tRNAs , and aminoacyl-tRNA synthetase , peptidyl transferases . Three steps : initiation, elongation, and termination . Posttranslational modifications happen

33. Initiation phase First, the small subunit of the ribosome and an initiator tRNA molecule assemble on the mRNA transcript. The small subunit of the ribosome has three binding sites : an amino acid site (A), a polypeptide site (P), and an exit site (E). The initiator tRNA molecule carrying the amino acid methionine binds to the AUG start codon of the mRNA transcript at the ribosome’s P site -become the first amino acid incorporated into the growing polypeptide chain.

34. Elongation phase First, the ribosome moves along the mRNA in the 5'-to-3'direction, which requires the elongation factor G, in a process called translocation. The tRNA that corresponds to the second codon can then bind to the A site , a step that requires elongation factors (in  E. coli , these are called EF- Tu and EF-Ts), as well as guanosine triphosphate (GTP) as an energy source for the process. Upon binding of the tRNA -amino acid complex in the A site, GTP is cleaved to form guanosine diphosphate (GDP), then released along with EF- Tu to be recycled by EF-Ts for the next round. Next,  peptide bonds between the now-adjacent first and second amino acids are formed through a  peptidyl transferase  activity .

35. After the peptide bond is formed, the ribosome shifts, or translocates , again, thus causing the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to pick up another amino acid. In addition, the A site is now empty and ready to receive the tRNA for the next codon . This process is repeated until all the codons in the mRNA have been read by tRNA molecules , and the nascent protein must be released from the mRNA and ribosome.

36. Termination of Translation There are three termination codons that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA. No tRNAs recognize these codons . Hence release factors , binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.

37. Post translational modifications to protein Translated p roteins undergo chemical modifications before becoming functional in different body cells Play a crucial role in generating the heterogeneity in proteins

38. Glycosylation : by the addition of carbohydrates, a process called glycosylation . It results in addition of a glycosyl group to either asparagine , hydroxylysine , serine, or threonine . Acetylation : the addition of an acetyl group, usually at the N-terminus of the protein. Alkylation : The addition of an alkyl group (e.g. methyl, ethyl ) Methylation : The addition of a methyl group, usually at lysine or arginine residues.

39. Phosphorylation : the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine Biotinylation : Acylation of conserved lysine residues with a biotin appendage. Glutamylation : Covalent linkage of glutamic acid residues to tubulin and some other proteins. Glycylation : Covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail of the amino acid sequence.

40. Isoprenylation : The addition of an isoprenoid group (e.g. farnesol and geranylgeraniol ). Lipoylation : The attachment of a lipoate functionality. Phosphopantetheinylation : The addition of a 4'-phosphopantetheinyl moiety to acyl carrier proteins Sulfation : The addition of a sulfate group to a tyrosine.

41. CONCLUSION Even while transcription is the primary mechanism for controlling gene expression, translation and its regulation is critical in both prokaryotic and eukaryotic cells. All elements of cell behaviour are ultimately regulated by these various controls on the levels and activities of intracellular proteins.

42. DNA mutations modify the mRNA sequence , which modifies the amino acid sequence. Mutations can shorten polypeptide chains by causing early translation termination. Alternatively, a mutation in the mRNA sequence affects the encoded amino acid. This amino acid variation affect protein function or folding.

43. PROKRYOTES AND EUKARYOTES

44. References Smith, C. M., Marks, A. D., Lieberman, M. A., Marks, D. B., & Marks, D. B. (2005). Marks’ basic medical biochemistry: A clinical approach. Philadelphia: Lippincott Williams & Wilkins. David Hames and Nigel Hooper (2005). Biochemistry and Genetics. Third ed. Taylor & Francis Group: New York. Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi: Jaypee Brothers Medical Publishers

45. https://byjus.com/biology/lac-operon-regulation-gene-expression https://www.khanacademy.org

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