DNA, Proteins and the Proteome

1. DNA, Proteinsand the Proteome Applications of molecular biology

2. Central dogma of molecular biology • The central dogma outlines the flow of information that is stored in a gene, transcribed into RNA and finally translated into protein. • The ultimate expression of this information is the phenotype of the organism.

3. Central dogma of molecular biology • DNA is self-replicating. • DNA is transcribed to produce mRNA. • mRNA is translated to produce protein.

4. Revision DNA and RNA • Both are polynucleotide chains but they have different functions and structures. • Three types of RNA – mRNA is messenger RNA. Proteins • Protein molecules carry out essential cellular functions and form the basis of many cell structures. • Virtually everything a cell is or does depends upon the proteins it contains.

5. The genetic code • The genetic codes consists of four nucleotides (A, C, G, T) and provides the instructions to make each of the 100,000+ proteins in the human body. • The code is read from 5’ to 3’ end of a DNA sequence and is usually written from left to right. • A group of three bases codes for one amino acid. • The genetic code codes for 20 amino acids and three STOP codons. • DNA code is copied (transcribed) to produce mRNA, and the order of amino acids in proteins is determined by the sequence of the three letter codes in mRNA.

7. The genetic code in action

8. Why the genetic code is special The genetic code is: • Universal • it is the same in all species. • Unambiguous • Each codon only codes for one amino acid. • Redundant • there is more than one codon for the same amino acid.

9. Why have DNA and RNA? • DNA is found in the nucleus and holds all the genetic information/instructions for the proteins produced in a cell. • If DNA ventured into the cytoplasm to give instructions for protein synthesis it would be vulnerable to damage from chemicals, UV radiation and other mutagens. • If DNA is damaged in any way, the coding sequence can change and a MUTATION will arise that will potentially influence the particular protein and perhaps the whole cell or organism. • RNA acts as a messenger. Damage to mRNA will not permanently affect function of the cell as the DNA template is undamaged.

10. Genes and proteins • All organisms contain the DNA and use the same arbitrary triplet code (the genetic code) to translate the nucleotide sequences of DNA into the amino acid sequences of proteins. • DNA is organised into segments called genes, which, by coding for the production of all proteins, determine the inherited characteristics of organisms. • A typical human cell may have about 20 000 different genes, but it can produce 200 000 different functional proteins. • One gene codes for one polypeptide, but this gene may produce many different functional proteins. Proteins can be altered by: • protease enzymes that cut polypeptides • chaperone proteins that bend them into different shapes • other enzymes that add cofactors, or carbohydrate chains, lipids or methyl groups.

11. Transcription and translation Transcription • Occurs in nucleus • Involves copying of DNA to mRNA Translation • mRNA leaves nucleus and travels to ribosomes in cytoplasm • Ribosome ‘reads’ mRNA and links amino acids to each other in sequence • Amino acids are carried to ribosome by tRNA molecules

13. Genomics and proteomics • An organism’s genome is all its genes. • The genome is the material that is passed on from generation to generation. It is the molecular store and carrier of information from cell to cell. • Genes are just templates, it is the proteins they produce that do the work in cells. • This has led to an interest in the products of the genome, the proteome, which is the entire protein complement of an organism.

14. Definitions for exam purposes Genome • an all inclusive word that describes the full set of genes that are present in the nucleus of a normal cell of a particular species. Proteome • all the protein products of the genome.

15. Importance of genomics • Genome projects involve working out the nucleotide sequence of all the genes of a species. • The availability of extensive genetic maps has increased the pace by which different disease genes are localized in the human genome. • Many new techniques developed during the human genome project have provided doctors with improved genetic diagnostics and predictive testing. • This is particularly true of diseases caused by single gene defects such as PKU, haemophilia and cystic fibrosis. Prenatal testing is available for many of these diseases and in some cases gene therapy options are being explored • Also allowed identification of susceptible areas of the genome that may be responsible for some disorders, such as diabetes, hypertension and certain forms of cancer that are more complicated and are caused by more than one genetic change.

16. Importance of proteomics • Genome projects have provided information about the primary sequences of the products of genes. • Proteomics opens the way for us to study genes at work—to understand proteins and how they interact in cells. • By understanding the links between genes, proteins and function, we will be able to: • develop more effective products and processes for agriculture and farming. • develop a better understanding of the causes of diseases and differing responses of individuals to those diseases, which should lead to better treatments. • Multidisciplinary teams are currently using proteomics to develop new tools to investigate the causes and diagnosis of infectious and genetic diseases and to develop specific proteomic medications, pharmaceuticals and vaccines.

17. The next step • After genomics and proteomics, the next ‘-omic’ may well be physiological genomics. • Physiological genomics examines the actual actions of proteins in living cells or organisms. • Physiological genomics is the ultimate test of any hypothesis with regard to the function of genes and proteins, and it can be used to evaluate their roles in living organisms as they live in their normal environments. • Physiological genomics can be as simple as monitoring the function of a single cell into which a SNP (single nucleotide polymorphism) has been inserted. It can also be as complex as the creation of a whole new animal, which has inherited the SNP as part of its own DNA sequence. • Such experiments are necessary to prove that particular SNPs truly are the cause of a cascade of events that lead to disease.

18. Designer Drugs • Drugs are chemicals that are given with the intention of affecting some aspect of the metabolic or psychological function of an organism. • Drugs can have different effects on different people. • The more precisely drugs can be tailored to an individual, the more effective they should be.

19. Factors to consider when designing drugs • Entering the cell • All chemicals must enter a cell via the cell membrane. • The manner in which this occurs is dependent on the chemical composition of the drug. • Action of drug • Bind to specific sites • Block or alter binding sites for other molecules. • Structure of drug and target molecule • Proteins have complex 3D shape • Need to ensure that active sites of drug correspond to binding sites of target

20. Examples of success • Relenza • Used to treat influenza • Targets neuraminidase protein of virus. This protein allows the exit of new virus particles from a cell, freeing them to infect other cells. • Gleevec • Used to treat chronic myeloid leukemia (CML), a cancer of white blood cells • Targets abnormal proteins that are fundamental to the cancer itself.

22. Other applications of molecular biology

23. Genetic Engineering • Genetic engineering allows scientists to pluck genes--segments of DNA--from one type of organism and to combine them with genes of a second organism. • In this way, relatively simple organisms such as bacteria or yeast can be induced to make quantities of human proteins, including interferons and interleukins. • They can also manufacture proteins from infectious agents, such as the hepatitis virus or the AIDS virus, for use in vaccines.

24. A plasmid (ring of DNA) is isolated from a bacterium An enzyme cuts the DNA at specific sites A gene for protein, taken from another cell, is cut with the same enzyme The recombinant plasmid is inserted back into the bacterium The new gene directs the bacterium to make a new protein product such as interferon The gene is inserted into the plasmid, where it fits exactly. This is recombinant DNA When the bacterium divides and replicates, it copies itself and the recombinant DNA Genetic Engineering

25. Understanding bacterial drug resistance • The development of drug resistance has become a huge problem in medicine, limiting the usefulness of many drugs. • Bacteria can resist antibiotics in a variety of ways: • Reduce intake of the drug • Alter the target molecule the drug attaches to • Increase elimination of the drug from the cell • Enzymatically deactivate the cell • If these resistant properties are present in members of the bacterial population, the bacteria possessing them will survive the treatment and their progeny will posses the same genes and therefore drug resistance. • Alternatively drug resistance can be transferred between bacteria in a process known as conjugation, in which small pieces of DNA are exchanged.

26. Antimicrobial resistance due to mutation

27. Transfer of resistance genes carried on plasmids through the process of conjugation

28. Managing AIDS • The severity and impact of the AIDS epidemic lead to the development of a number of drugs that can prolong and improve the quality of life • Many of these treatments have serious side-effects, however, in many cases it can reduce levels of HIV in the blood, halt the progress of the disease and allow recovery of immune function • Different drugs target different stages of the HIV life cycle Example: • AZT targets viral reverse transcriptase and has had a significant effect on perinatal HIV infection (mother to infant). • Perinatal HIV infection accounts for 5-10% of transmission worldwide. • In the absence of AZT, 25% of children born to HIV positive mothers become infected in utero, during delivery, or by breast-feeding. When AZT treatment is available this drops to 3%.

29. Drugs to treat HIV • Attachment blockers: block cell receptors preventing the HIV from attaching • Entry inhibitors: prevent the virus from fusing to and entering the cell • Reverse transcriptase inhibitors: prevent the synthesis of DNA from viral RNA • Integrase inhibitors: prevent HIV DNA from integrating into the cell’s DNA • Protease inhibitors: prevent newly formed viral RNA from assembling the proteins of the viral coat.