PTM and Biotechnology (1) PDF

Biotechnology & Genetic Engineering Recombinant DNA Technology Post Translational Modification (PTM) 2 3 (1) Trimming 🠶 They become activated 🠶 Many proteins are through cleavage when 🠶 E.g: Conversi...

Biotechnology & Genetic Engineering Recombinant DNA Technology Post Translational Modification (PTM) 2 3 (1) Trimming 🠶 They become activated 🠶 Many proteins are through cleavage when 🠶 E.g: Conversion of made in the form of large they reach their proper inactive Pepsinogen into precursor molecules which sites of action, by the active pepsin, by the action are not functionally active action of other enzymes / of HCl. (called Zymogens). non-enzymes factors. 4 (2) Covalent Modification (i) Phosphorylation 🠶 Addition of phosphate group to amino acid residues of proteins (Ser, Thr, Tyr). 🠶 Catalyzed by family of enzymes Protein Kinases.  (ii) Glycosylation 5 🠶 Addition of carbohydrate chain to protein. 🠶 Occurs only in those proteins which have to become the part of cell membrane. (iii)Hydroxylation 🠶 Collagen is hydroxylated in ER. (iv)Others 🠶 Carboxylation. 🠶 Biotinylation 🠶 Farnisylation (attachment of lipid groups)  (3) Protein folding 6 🠶 Important for initiation of their functional activity. 🠶 Folding may be 🠶 Spontaneously induced 🠶 Facilitated by other proteins (called Chaperons). (4) Protein degradation 🠶 Unfolded and defective proteins are degraded by enzymes called Post Transcriptional Modification ▪ In bacteria, RNA transcripts are ready to act as messenger RNAs and get translated into proteins right away. ▪ In humans and other eukaryotes, a freshly made RNA transcript is called a pre- mRNA and has to go through some processing steps to become a mature messenger RNA (mRNA) that can be translated into a protein. These include: Addition of a 5' cap to the beginning of the RNA Addition of a poly-A tail (tail of A nucleotides) to the end of the RNA Chopping out of introns, or "junk" sequences, and pasting together of the remaining, good sequences (exons) Capping and Tailing ▪ Both ends of a pre-mRNA are modified by the addition of chemical groups. ▪ The group at the beginning (5' end) is called a cap, while the group at the end (3' end) is called a tail. ▪ Both the cap and the tail protect the transcript and help it get exported from the nucleus and translated on the ribosomes ▪ The 5’ cap is added to the first nucleotide in the transcript during transcription. The cap is a modified guanine (G) nucleotide, and it protects the transcript from being broken down. It also helps the ribosome attach to the mRNA and start reading it to make a protein. ▪ The poly-A tail is on the 3' end of the pre-mRNA and consists of a long string of A nucleotides ▪ When a sequence called a polyadenylation signal shows up in an RNA molecule during transcription, an enzyme chops the RNA in two at that site. ▪ Another enzyme adds about 100-200 adenine (A) nucleotides to the cut end, forming a poly-A tail. The tail makes the transcript more stable and helps it get exported from the nucleus to the cytosol.  Splicing ▪ In RNA splicing, specific parts of the pre-mRNA, called introns are recognized and removed by a protein-and-RNA complex called the spliceosome. ▪ Introns can be viewed as "junk" sequences that must be cut out so the RNA molecule can be assembled. ▪ The pieces of the RNA that are not chopped out are called exons. The exons are pasted together by the spliceosome to make the final, mature mRNA that is shipped out of the nucleus. ▪ only the exons of a gene that encode a protein ▪ If the spliceosome fails to remove an intron, an mRNA with extra "junk" in it will be made, and a wrong protein will get produced during translation. Alternative splicing ▪ splicing does allow for a process called alternative splicing, in which more than one mRNA can be made from the same gene. ▪ Through alternative splicing, eukaryotes can encode more different proteins than theyhave genes in DNA. ▪ In alternative splicing, one pre- mRNA may be spliced in either of two (or sometimes many more than two!) different ways. ▪ This results in three different mature mRNAs, each of which translates into a protein with a different structure. ▪ Modification in nucleoside: ▪ Methylferases, deaminases and dehydrogenases may methylate, deaminate or reduce the bases into the ‘minor’ bases, ▪ e.g. 5 methylcytosine, N6 -methyladenine, hypoxanthine, dihydrouracil, etc. ▪ Uridine may be converted into pseudouridine. ▪ Ligations and cleavages of nucleotides: ▪ The gene contains exons and introns. ▪ The introns need to be separated out and exons must be joined as they are actual amino acid coding sequences. ▪ Specific nucleases and ligases bring about this function. ▪ These changed an RNA into functional m-RNA. ▪ Additional nucleotides may be added at the end of RNA transcript, e.g. 7 methyl GTP cap is added at 5’-end while poly A-tail is added at the 3’-end Introduction to Biotechnology and Genetic Engineering 7  BIOTECHNOLOGY 8 🠶 Biotechnology is any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use. Genetic Engineering 🠶 Genetic Engineering is the direct manipulation of an organism’s genome using biotechnology. 🠶 It is therefore a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. 9 Applications Medicine 🠶 Drug development (e.g: insulin) 🠶 Pharmacogenomics 🠶 Genetic testing 🠶 Diagnostics Agriculture 🠶 Genetically modified crops (Bacillus thurengiensis utilization) 🠶 Genetically modified foods / live stocks Industries 🠶 Brewing 🠶 Renewable raw material Environmental 🠶 Oil eating bacteria 10 Important Techniques DNA Cloning 🠶 A DNA molecule from a single living cell is used to make a large population of cells containing identical DNA molecules. 🠶 It is used to assemble recombinant DNA molecules, and to direct their replication within host organisms. 🠶 Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for multiplying (replicating) the recombinant DNA. Blotting 🠶 A technique used for the detection of macromolecules. 🠶 Southern Blotting: Detection of DNA 🠶 Northern Blotting: Detection of RNA 🠶 Western Blotting: Detection of Proteins 11 Important Techniques Polymerase Chain Reaction (PCR) 🠶 A method for amplifying a selected DNA sequence. 🠶 PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. 🠶 It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. 🠶 The method can be used to amplify DNA sequences from any source— bacterial, viral, plant, or animal. 12 Recombinant DNA (rDNA) Technology 13 14 rDNA 🠶 Recombinant DNA (rDNA) molecules are DNA molecules formed through genetic engineering to bring together genetic material from multiple sources, thereby creating sequences that would not otherwise be found naturally, in order to produce a modified gene product of interest. 🠶 Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. 15 Genetically Engineered Glowing Plants containing ‘luc’ Gene From Firefly 16 Basic Working Principle 🠶 Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. 🠶 That alone, however, will not do much. 🠶 Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced and then expressed. This is known as DNA cloning. 17 18 Basic Working Principle ▪ The steps include ▪ isolating of the target gene and the vector, ▪ specific cutting of DNA at defined sites, ▪ joining of DNA fragments, ▪ Transferring rDNA to host cell, ▪ cloning, 19 Tools / Requirements ▪ The various biological tools used to bring about genetic manipulations are: ▪ A. Enzymes ▪ B. Passenger DNA: Foreign DNA ▪ C. Vector or vehicle DNA: Enzymes: ▪ The various enzymes which may be required to be used are: ▪ Restriction endonucleases: To cut DNA chains at specific locations (called as “chemical knife”) ▪ Exonucleases: To cut DNA at 5' terminus- helps in DNA repair ▪ Endonucleases: To cut in the interior to produce “nicks” ▪ Reverse transcriptase ▪ DNA polymerases ▪ DNA ligase (T4 ligase) 20 Restriction Endonuclease 🠶 Also called ‘Biological Scissors’. 🠶 Cleaves double-stranded (ds) DNA into smaller, more manageable fragments, so that they can be joined with the vector DNA (carrier DNA). 🠶 Gene of interest can be isolated from the DNA by treating the DNA molecule with restriction enzymes.  🠶 Restriction endonucleases cuts DNA with sticky ends. 21 🠶 In this way sticky ends of both DNA molecules (i.e: vector DNA and target DNA can be joined together by complementary base pairing. 🠶 Sugar phosphate backbone is completed by DNA Ligase. ▪ Each restriction endonuclease enzyme recognises and cleaves a specific double-stranded DNA sequence that is 4 to 7 bp long. ▪ These DNA cuts result in: ▪ Blunt ends (by Hpa I) ▪ Sticky ends (also called staggered or cohesive ends) by Bam HI or Eco R I. ▪ Sticky ends are particularly useful in preparation of chimeric or hybrid DNA molecule. Pharmaceutical Applications 22 23 Recombinant Pharmaceutical Products ▪ 🠶 Large-scale production of human hormones and other proteins with ▪ therapeutic uses ▪ 🠶 Production of vaccines ▪ 🠶 A number of therapeutic gene products are now produced ▪ commercially from cloned genes ▪ 🠶 insulin, ▪ 🠶 the interleukins, ▪ 🠶 interferons, ▪ 🠶 growth hormones, ▪ 🠶 erythropoietin, and ▪ 🠶 coagulation factor VIII— Enzyme Ribonuclease (RNase) 26  Introduction 27 ▪ 🠶 Ribonuclease (RNase) is a type of nuclease enzyme class that catalyzes the degradation of RNA by cleaving it into smaller components (mono- nucleotides) ▪ Functions ▪ 🠶 cleaning of cellular RNA that is no longer required. ▪ 🠶 Protection against certain RNA viruses. 28 Mechanism of Action 🠶 Catalyzes the cleavage of RNA strand by Hydrolysis reaction. 🠶 Hydrolyze RNA by cutting the P-O ester bond attached to ribose 5’ carbon.  29 RNase RNase target Important Concepts of Gene Expression Genetic Switch ▪ Gene switching is defined as the reversible on/off switching of gene expression or the switching of expression between two alleles of a gene. ▪ It involves transient changes in gene expression in response to environmental variations at the level of transcription, translation, or post- translation, ultimately pre- adapting cells for new environmental conditions through alterations in gene expression. Operon ▪ The concept of operon was introduced by Jacob and Monod in 1961. ▪ Operon is defined as a segment of a DNA strand consisting of: ▪ Structural genes: A cluster of several structural genes, which carries the codons which can be translated into protein. ▪ Operator gene: One operator gene which has an overall control over the process of translation. ▪ Regulator gene: A third gene called regulator gene is located sometimes at a distance from the operator gene on the same DNA strand. Regulator gene transcribes m-RNA which synthesises repressor protein molecules which regulate the transcription. ▪ P site (promoter site): It is situated between operator gene and regulator gene. Inducers, Repression and Derepression ▪ The “lac operon” is an inducible catabolic operon of E. coli. It carries three structural genes: ‘Z’, ‘Y’ and ‘A’ ▪ Has a single promoter (transcribed as a single mRNA) encode proteins that allow the bacteria to use lactose as an energy source. ▪ The promoter is the binding site for RNA polymerase, the enzyme that performs transcription. ▪ The operator is a negative regulatory site bound by the lac repressor protein. ▪ The operator overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot bind to the promoter and start transcription. ▪ The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter. ▪ The lac repressor is a protein that represses (inhibits) transcription of the lac operon. ▪ When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription ▪ However, when lactose is present, the lac repressor loses its ability to bind DNA. clearing the way for RNA polymerase to transcribe the operon. ▪ This change in the lac repressor is caused by the small molecule allolactose, an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. ▪ Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or operon. ▪ The lac operon is considered an inducible operon because it is usually turned off (repressed), but can be turned on in the presence of the inducer allolactose. ▪ Fusion genes are formed from two distinct genes as a result of chromosomal rearrangements, deletions, inversions or translocations. In molecular biology, fusion genes may also be created deliberately, as in proteins that are fused to fluorescent proteins to enable their microscopic visualization. ▪ specificity involved in the control of transcription requires that the regulatory proteins bind with high affinity to the correct region of DNA. 30 REFERENCES 🠶 Lippincott’s Illustrated Reviews: Biochemistry Fifth Edition 🠶 Textbook of Medical Edition. Biochemistry, MN Chatterjea, 8th 🠶 Lehninger PRINCIPLES OF BIOCHEMISTRY Fourth Edition