MB 206 Microbial Biotechnology

1. MB 206 Microbial Biotechnology MB206 May-Aug 09 Angelia Teo

2. CHAPTER 1.1 NUCLEIC ACID

3. A Comparison of DNA and RNA

4. Messenger RNA (mRNA) Ribosome: contains ribosomal RNA (rRNA) catalytic site large subunit 1 2 tRNA/amino acid binding sites small subunit Transfer RNA (tRNA) attached amino acid anticodon

5. Central Dogma of Molecular Biology How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? • DNA codes for RNA production. • RNA codes for protein production. • Protein does not code protein, RNA • or DNA production. • The end. • Or in the words of Francis Crick: Once information has passed into • protein, it cannot get out again!

6. Revision of the "Central Dogma" • CAN go back from RNA to DNA (reverse transcriptase) • RNA can also make copies of itself (RNA polymerase) • Still NOT possible from Proteins back to RNA or DNA • Not known mechanisms for proteins making copies of themselves.

7. Gene Expression • Expression of genetic determinants in bacteria involves the • unidirectional flow of information from DNA to RNA to • protein. • Two processes involved are transcription and translation.

8. Transcription & Translation Prokaryotic vs Eukaryotic cells In a prokaryotic cell, which does not contain a nucleus, this process happens at the same time. In Eukaryotic cells, occur at different cell compartments. Prokaryotic cell Eukaryotic cell

9. Transcription • The DNA-directed synthesis of RNA is called transcription. • Transcription produces RNA molecules that are complimentary copies of one strand of DNA. • Only one of the dsDNA strands can serve as template for synthesis of a specific mRNA molecule. • mRNAs transmit information from DNA, and each mRNA in bacteria function as a template for synthesis of one or more specific proteins.

10. Translation • Initiated at an AUG codon for methionine. • Codons are translated sequentially in mRNA from 5' to 3'. • The corresponding polypeptide chain / protein is assembled from the amino terminus to carboxy terminus. • The sequence of amino acids in the polypeptide is, therefore, co-linear with the sequence of nucleotides in the mRNA and the corresponding gene.

11. The Genetic code The "universal" genetic code employed by most organisms is a triplet code and it determines how the nucleotides in mRNA specify the amino acids in the polypeptide. • 61 of 64 possible trinucleotides (codons) encode specific amino acids. • 3 remaining codons (UAG, UAA or UGA) code for termination of translation (nonsense codons = do not specify any amino acids) • Exceptions: • UGA as a tryptophan codon in some species of Mycoplasma and in mitochondrial DNA. • Few codon differences in mitochondrial DNAs from yeasts, Drosophila, and mammals.

12. Gene expression occurs in 2 steps: Transcriptionof the information encoded in DNA into a molecule of RNA Translation of the information encoded in mRNA into a defined sequence of amino acids in a protein.

13. Organization of bacterial chromosome Prokaryotic DNA replicate, transcription & translation Module 1-1Bacterial Genetics by Angelia Teo (Jan 09)

14. Prokaryote • the genome of prokaryotes is not in a separate compartment, haploid. Single chromosome: it is located in the cytoplasm (although sometimes confined to a particular region called a “nucleoid”). Prokaryotes contain no membrane-bound organelles; their only membrane is the membrane that separates the cell form the outside world. Nearly all prokaryotes are unicellular. Eukaryotes are defined as having their genetic material enclosed in a membrane-bound nucleus, separate from the cytoplasm. In addition, eukaryotes have other membrane-bound organelles such as mitochondria, lysosomes, and endoplasmic reticulum. almost all multicellular organisms are eukaryotes. by Angelia Teo (Jan 09)

15. Prokaryote cond.. • Prokaryotes are haploid, and they contain a single circular chromosome. In addition, prokaryotes often contain small circular DNA molecules called “plasmids”, that confer useful properties such as drug resistance. Only circular DNA molecules in prokaryotes can replicate. Eukaryotes are often diploid, and eukaryotes have linear chromosomes, usually more than 1. by Angelia Teo (Jan 09)

16. Prokaryote cond.. • In prokaryotes, translation is coupled to transcription: translation of the new RNA molecule starts before transcription is finished. In eukaryotes, transcription of genes in RNA occurs in the nucleus, and translation of that RNA into protein occurs in the cytoplasm. The two processes are separated from each other. by Angelia Teo (Jan 09)

17. Bacteria • Bacteria review • one-celled organisms • prokaryotes • reproduce by mitosis • binary fission • rapid growth • generation every ~20 minutes • 108 (100 million) colony overnight! • dominant form of life on Earth • incredibly diverse

18. Bacterial genome • Single circular chromosome • haploid • naked DNA • no histone proteins • ~4 million base pairs • ~4300 genes • 1/1000 DNA in eukaryote

19. No nucleus! • No nuclear membrane • chromosome in cytoplasm • transcription & translation are coupled together • no processing of mRNA • no introns • but Central Dogma still applies • use same genetic code

20. Bacterial Chromosome • Molecules of double-stranded DNA • Usually circular • Tend to be shorter • Contains a few thousand unique genes • Mostly structural genes • Single origin of replication

21. Bacterial Chromosome cond.. • The bacterial chromosome is found in region called the nucleoid (not membrane-bounded- so the DNA is in direct contact with the cytoplasm) by Angelia Teo (Jan 09)

22. Bacterial Chromosome cond.. Bacterial Genome is haploid, single chromosome • The circularity of the bacterial chromosome was elegantly demonstrated by electron microscopy in both Gram negative bacteria (such as Escherichia coli) and Gram positive bacteria (such as Bacillus subtilis). • Bacterial plasmids were also shown to be circular. • Linear chromosomes found in Gram-positive Borrelia & Streptomyces. Not all bacteria have a single circular chromosome: some bacteria have multiple circular chromosomes, and many bacteria have linear chromosomes and linear plasmids. by Angelia Teo (Jan 09)

23. The Operon Model The operon model of prokaryotic gene regulation was proposed by Fancois Jacob and Jacques Monod. Groups of genes coding for related proteins are arranged in units known as operons. An operon consists of an operator, promoter, regulator, and structural genes. The regulator gene codes for a repressor protein that binds to the operator, obstructing the promoter (thus, transcription) of the structural genes. The regulator does not have to be adjacent to other genes in the operon. If the repressor protein is removed, transcription may occur. by Angelia Teo (Jan 09)

24. The Operon Model Operons are either inducible or repressible according to the control mechanism. Seventy-five different operons controlling 250 structural genes have been identified for E. coli. Both repression and induction are examples of negative control since the repressor proteins turn off transcription. by Angelia Teo (Jan 09)

25. The Operon Model by Angelia Teo (Jan 09)

26. Extra-chromosomal Elements • DNA molecules that replicate as discrete genetic units in bacteria are called replicons. • Extrachromosomal replicons: - bacteriophages - plasmids (non-essential replicons) • These determine resistance to antimicrobial agents or production of virulence factors. by Angelia Teo (Jan 09)

27. Regulation of Gene Expression in Bacteria MB 206 : Module 1 - B Angelia Teo Jan 09

28. Regulation of Gene Expression • A cell contains the entire genome of an organism– ALL the DNA. • Gene expression= transcribing and translating the gene • Regulation allows an organism to selectively transcribe (and then translate) only the genes it needs to. • Genes expressed depend on • the type of cell • the particular needs of the cell at that time.

29. How Are Genes Regulated? • Genes located in coherent packages called operons • operons has 4 parts • regulatory gene - controls timing or rate of transcription • promoter - starting point • operator - controls access to the promoter by RNA polymerase • structural genes • NOTE = operons regulated as units Angelia Teo Jan 09

30. Most genes are not expressed at a particular time • Not all of the genes in a bacteria will be expressed at the same time. • Even in some of the smallest bacteria, about 500 different genes exists • Of the 4279 genes in E. coli , only about 2600 (~60%) are expressed in standard laboratory conditions. • Only about 350 genes are expressed at more than 100 copies (i.e. molecules!) per cell, making up 90% of the total protein. Angelia Teo Jan 09

31. Possible target in control of gene expression

32. MB 206 : Module 2 - C Mutations & Selections

33. Mutation Role of Mutation

34. How Do Mutations in DNA Affect the Function of Genes? • Mutations result from Nucleotide Substitutions, Insertions, or Deletions • Mutations may have a variety of effects on protein structure and function • Mutations provide the raw material for evolution Chapter 10.

35. MUTATION • Process of mutation may result in a gene coding for a new protein • Mutations are not good or bad, they do provide the raw material of change. • There are many different types of mutations. Chapter 10.

36. Type of Mutation Nucleotide substitution can result in either a change or no change in amino acid of the codon.

37. MUTATION • Point Mutation - change in one nucleotide. • Insertion - new nucleotide pairs are inserted into DNA molecule • Deletion - nucleotide pairs are removed from DNA molecule Chapter 10.

38. Different Types of Mutations

39. Plasmid Plasmid – small, circular, extrachromosomal DNA which replicates independently of host chromosomal DNA • First plasmid described was discovered in Japan in Shigella species during an outbreak of dysentery in the early 1940‘s • 3 main components: • Origin of replication • Selectable marker • Restriction enzyme site(s) • Enzymes that cut at specific sequence on DNA

40. Plasmids Content • Replication factors • Genes

41. Ori Region • Ori, actual site of replication • Proteins that assist in replication (varies) • Recognition sequences for control factors • The ori determines the Range

42. Plasmids • Discrete, extrachromosomal genetic elements in bacteria • Usually much smaller than bacterial chromosome • Size varies from < 5kb to > 100 kbp • Mostly supercoiled, circular, ds DNA molecules • Replicate independently of the chromosome • Exist in multiple copies in bacterial (the average number of plasmid per bacterial is called copy number). • Usually encode traits that are non-essential for bacterial viability.

43. Plasmid is an ideal structure for genetic engineering because • Simple in structure • Easy to extract & isolate in the lab • Easy for genetic manipulation & transformed back into bacteria • Contains genetic information which can be used by the bacteria • Most plasmid present in high copy number • Plasmid codes for antibiotic resistant gene eg. Ampicillin, Apr or Tetracyclin Tcr - selection of bacteria with transformed plasmid. • Non-essential for bacteria’s growth, thus possible to manipulate plasmid DNA without affecting bacteria growth.

44. Exchange of Genetic Information in bacteria • Medically important - rapid emergence and dissemination of antibiotic resistance plasmids - flagellar phase variation (eg. Salmonella) - antigenic variation of surface antigens (eg. Neisseria & Borrelia) • Sexual processes in bacteria involve transfer of genetic information from a donor to a recipient, results in: - substitution of donor alleles for recipient alleles - addition of donor genetic elements to the recipient genome. • 3 major types of genetic transfer found in bacteria: a) Transformation b) Transduction c) Conjugation In all three cases, recombination between donor and recipient DNA result in formation of stable recombinant genomes        

45. Types of transfers: • Non-transmissible = cannot initiate contact with recipient or transfer DNAConjugative = can initiate contact with recipient bacteriumMobilizable = can prepare its DNA for transferSelf-transmissible = is both conjugative & mobilizable • 4 stages of plasmid transfer: a) Effective contact b) Mobilization - preparation for DNA transfer c) DNA transfer d) Formation of F in recipient • Donation - a conjugative plasmid (F) can provide conjugative function to a mobilizable plasmid (eg. ColE1) such that both plasmids can be transferred. • Plasmid conduction - a self-transmissible plasmid (F) can recombine with a non-mobilizable plasmid and transfer the co-integrate.

46. Fin genes & plasmid transfer • fin genes - fertility inhibition - codes for repressor that prevents transcription of genes required for transfer. • F plasmid has 1 fin gene so transfer system is always ‘ON’ • R plasmid has 2 fin genes so cannot always transfer. - in new recipients (repressor is absent) so transfer can occur soon after receiving the R plasmid but after time (when repressor is made) transfer can't occur

47. (a) Bacterial Transformation • introducing DNA from donor to recipient • result in uptake and integration of fragments of donor DNA into recipient genome. • produce stable hybrid progeny. • is most likely to occur when the donor and recipient bacteria the same or closely related species.

48. Bacteria transformation in the lab "Transformation" is simply the process where bacteria manage to "uptake" a piece of external DNA.   Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell.

49. Bacteria Transduction • Bacteriophage infect donor bacterium • form rare abnormal bacteriophage particles contain DNA from donor bacteria. • abnormal bacteriophageinfect recipient bacteria & inject DNA into recipient • donor DNA integrated / recombined into recipient DNA resulting in transduced bacterium.

50. Bacterial Conjugation • Transfer of DNA between 2 bacteria in contact with each other • Contact between donor and recipient (initiated by sex pili) • DNA transfer through a conjugation bridge • Mediated by a plasmid • Called an F-factor (fertility factor) or conjugative plasmid