Microbial Genetics

1. Microbial Genetics Chapter 9

2. transmission of biological traits from parent to offspring • expression & variation of those traits • structure & function of genetic material • how this material changes Genetics – the study of heredity

3. Levels of genetic study

4. Genetic structure/function – of DNA, chromosomes, genes and genomes; also including size and arrangement both prokaryotes and eukaryotes. • Mechanisms of replication, transcription and translation including enzymes for proks., euks.andviruses. • Gene regulation: inducible vs. repressible operons (Prokaryotes only) • Mutations – types, causes and effects • Recombination – conjugation, transformation. • Famous names in the history of genetics What you need to know about microbial genetics…

5. Genetic structure/function • ____________ – sum total of genetic material of an organism (chromosomes + mitochondria/chloroplasts and/or plasmids) • genome of cells – DNA • genome of viruses – DNA or RNA • ____________– length of DNA containing genes • ____________ -fundamental unit of heredity responsible for a given trait • site on the chromosome that provides information for a certain cell function (____________) • segment of DNA that contains the necessary code to make a protein or RNA molecule Levels of structure & function of the genome

6. Genetic structure/function Locations and forms of genomes

7. Genetic structure/function • smallest virus – 4-5 genes • E. coli – single chromosome containing 4,288 genes; 1 mm; 1,000X longer than cell (~4.5 Mbp) • Human cell – 46 chromosomes containing 31,000 genes; 6 feet; 180,000X longer than cell Genomes vary in size

8. Genetic structure/function • Prokaryotes: coiled into tight bundle by gyrase (a topoisomerase) • Eukaryotes: 1. wound around histone proteins to form nucleosomes 2. nucleosomes condense, coil into chromatin fibers 3. Chromatin supercoils and condenses into __________________ Genome packaging

9. Genetic structure/function

10. Genetic structure/function • Nucleic acids are made of nucleotides (polymer, monomer) • each nucleotide consists of 3 parts • a 5 carbon _____________(deoxyribose or ribose) • a __________________group • a nitrogenous base (adenine, thymine, cytosine, guanine, and uracil) • Purines: A + G; Pyrimidines: C, U, T Nucleic acid structure (RNA, DNA)

11. Genetic structure/function • 2 strands twisted into a double helix • sugar -phosphate backbone • nitrogenous bases form steps in ladder (inside helix) • constancy of base pairing (purines pair with pyrimidines) • _______________ with 2 hydrogen bonds (RNA – A with U) • __________________ with 3 hydrogen bonds • antiparallel strands __________________ • each strand provides a template for the exact copying of a new strand • order of bases constitutes the DNA code DNA structure

12. Genetic structure/function

13. Genetic structure/function

14. Genetic structure/function Torsion in helix and “steps” of nucleotides results in major and minor grooves in helix

15. Genetic structure/function • Maintenance of code during reproduction. Constancy of base pairing guarantees that the code will be retained. • Providing variety. Order of basesresponsible for unique qualities of each organism (gene sequence) Possible arrangement of nucleotides is nearly infinite: 4n where n=#nucleotides. So for a 1000 bp gene, 4n combinations = 41000 or 1.5 x 10602 !! Significance of DNA structure

16. Genetics - History • __________________________ – 1944 – showed DNA was the molecule carrying the blueprint for life. Won Nobel prize. • Erwin Chargaff – components of DNA • Maurice Wilkins and ______________________– XRay crystallography gave clue to double helix structure • Structural model: credit (Nobel prize) goes to _________________________________- 1953 History of DNA structure

17. Genetic mechanisms -Replication • Due to base pairing, each strand serves as template for the synthesis of a new strand • DNA replication is ________________because each chromosome ends up with one new strand of DNA and one old strand. DNA – A with T, G with C – so one strand can be template for its complement strand DNA replication

18. Genetic mechanisms Genetic mechanisms -Replication Semi-conservative replication of DNA

19. Genetic mechanisms -Replication Steps: • Begins at an origin of replication • ___________unwinds and unzips the DNA double helix (replication fork) • Strands are kept separate by SSBP (single strand binding proteins) • An RNA ___________is synthesized (primase) and “primes” (DNA polymerase III cannot initiate synthesis on its own) DNA replication (bacteria)

20. Genetic mechanisms -Replication • DNA polymerase III adds nucleotides in a 5’ to 3’ direction – works on both strands at once (see movie!) • Leading strand – synthesized continuously in 5’ to 3’ direction • Lagging strand – synthesized 5’ to 3’ in short segments (Okazakifragments); • overall direction of DNA pol III movement is 3’ to 5’ • ___________– removes primers (RNA) and replaces with DNA • ___________– fills in gaps/nicks Replication (cont’d)

21. Genetic mechanisms -Replication Enzymes involved in DNA replication (short list)

22. Genetic mechanisms -Replication (origin is A/T rich, easy to separate) Bacterial replicon SEE THE TWO MOVIES ON THE WEB SITE!!

23. Genetic mechanisms -Replication • Separation of daughter molecules occurs via a “nick” which is then repaired • Two completed molecules will go to daughter cells (binary fission)

24. Genetic mechanisms -Replication • Eukaryotes – similar to this but there are thousands of replicons acting simultaneously (replication bubbles) • Rolling circle replication – small circular genetic material (plasmids) Other types of replication

25. Flow of genetic information

26. What are the products that genes encode? • Structural genes – code for proteins • Genes that code for RNA (they are not translated!) • Regulatory genes that control the expression of other genes • ___________– is an organisms genetic makeup • ___________– is the physical trait that results from the expression of the organism’s genes • How are genes expressed? • transcription and translation Flow of genetic information

27. Genetic mechanisms -Gene expression • ________________– DNA template is used to synthesize RNA (transcript) • ______________________is the enzyme responsible • ________________–making a protein using the information provided by messenger RNA (mRNA) – involves decoding the mRNA • occurs on ribosomes, and involves tRNA and amino acids • Proteins – end product of gene expression • Please view transcription + translation movies on the web page Gene expression

28. Genetic mechanisms -Gene expression • Each triplet of nucleotides (codon) specifies a particular amino acid. • structure  function A protein’s primary structure determines its shape & function. • Proteins determine phenotype. Living things are what their proteins make them. • DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them. DNA-protein relationship

29. Genetic mechanisms -Gene expression DNA-protein relationship

30. Genetic mechanisms -Gene expression • Three types: • messenger RNA (mRNA) • transfer RNA (tRNA) • ribosomal RNA (rRNA) • How does RNA differ from DNA? • Uses Uracil (U) instead of Thymine (T) • Single stranded (except in some viruses) • Ribose is the sugar RNA

32. Genetic mechanisms -Gene expression DNA Transcription RNA polymerase RNA Translation ribosomes PROTEINS

33. Genetic mechanisms -Gene expression • RNA polymerase binds to promoter region upstream of the gene • RNA polymerase adds nucleotides complementary to the template strand of a segment of DNA in the 5’ to 3’ direction (downstream of promoter) • Uracil is placed as adenine’s complement (U with A) • At termination, RNA polymerase recognizes signals and releases the transcript • 100-1,200 bases long Transcription - steps

34. Genetic mechanisms -Gene expression Transcription

35. Genetic mechanisms -Gene expression DNA Transcription RNA polymerase RNA Translation ribosomes PROTEINS

36. Genetic mechanisms -Gene expression • Ribosomes assemble on the ___________of a mRNA transcript • Ribosome scans the mRNA until it reaches the start codon, usually _______(met) • A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome & binds to the mRNA • mRNA triplet code is translated into amino acids (elongation) Translation - intiation

37. Genetic mechanisms -Gene expression Translation

38. Genetic mechanisms -Gene expression

39. Genetic mechanisms -Gene expression

40. Genetic mechanisms -Gene expression • A second tRNA with the complementary anticodon fills the A site • A peptide bond is formed • The first tRNA is released and the ribosome slides down to the next codon (5’3’ reading frame = triplet). • Another tRNA fills the A site & a peptide bond is formed. • This process continues until a stop codon is encountered. Translation elongation

42. Genetic mechanisms -Gene expression • Termination (________) codons – UAA, UAG, and UGA – are codons for which there is no corresponding tRNA. • When this codon is reached, the ribosome falls off and the last tRNA is removed from the polypeptide. Translation termination

43. Polyribosomal complex = transcription and multiple translation simultaneously (bacteria)

44. Genetic mechanisms -Gene expression • Do not occur simultaneously. Transcription occurs in the nucleus and translation occurs in the cytoplasm. • Eucaryotic start codon is AUG, but it does not use formyl-methionine. • Eucaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many (operon). • Eucaryotic DNA contains introns – intervening sequences of noncoding DNA- which have to be spliced out of the final mRNA transcript. Eucaryotic transcription & translation differs from procaryotic

45. Genetic mechanisms -Gene expression Split gene of eucaryotes

46. Genetic mechanisms -Gene expression • Genes encoding for RNAs such as tRNA, rRNA, RNA primers (used in DNA replication) • These RNAs have 2ndary structure like proteins but function as RNA Some transcribed genes aren’t translated

47. Regulation of protein synthesis & metabolism

48. Gene regulation • a coordinated set of genes, all of which are regulated as a single unit. Found in prokaryotes. • 2 types – based on regulation • ________________– operon is turned ON by substrate: catabolic operons- enzymes needed to metabolize a nutrient are produced when needed • ________________– operon is turned OFF by the product synthesized; anabolic operon –enzymes used to synthesize an amino acid stop being produced when enough is made Operons

49. Gene regulation Made of 3 segments: • ________________gene that codes for ________________ • ___________locus- composed of promoter and operator • ___________locus- made of 3 genes each coding for an enzyme needed to catabolize lactose – b-galactosidase – hydolyzes lactose (gal and glu) permease - brings lactose across cell membrane b-galactoside transacetylase – uncertain function Lactose operon: inducible operon

50. Gene regulation • ________________ • In the absence of lactose the repressor binds with the operator locus and blocks transcription of downstream structural genes • Lactose ____________________________ • Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. RNA polymerase can bind to the promoter. Structural genes are transcribed. Lac operon: inducible