Scribe.Lecture 23.docx

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University of Toronto St. George
Cell and Systems Biology
William Navarre

Lecture 23 1 1. bacterial competence, bacteria are using energy to take up DNA in order for them to take up DNA as food or for biofilm formation 2. organisms are the vessels for genes, genes are the selfish elements that are driving organisms’ behaviour, they are trying to propagate, everything we do is making our genes survive, genes are using us to spread themselves 3. here we will talk about genetic elements that do things independent of the cell to spread to the pop, selfish gene 4. selfish genes can’t be entirely selfish bc they can’t kill the host, they must cooperate w the preexisting genes of the DNA it is inserting into 2 5. transduction and conjugation are examples of selfish genes – genes are mobilizing themselves against the will of the recipient host 1. in conjugation, a donor bacterium/plasmid encodes its own machinery and injects itself into an uninfected cell 2. in transduction, the selfish phage, genes that accidentally piggyback into cell 1. some genes are aware they are infecting phage and block phage itself from putting its own genes 3 6. plasmids are found in a number of species 1. the diff strains of E. coli, some have plasmids, some don’t 7. there is evidence of extra chromosomal elements that confer extra functionality to the host 8. under normal (nonstress) conditions, you can get rid of the plasmids and the bacterial chromosome will do fine 4 9. plasmids are USUALLY circular, small, supercoiled DNA molecules 10. each plasmid can exist as a single copy or multiple copies (plasmid copy number) 11. they have their own replication origin are autonomously replicated, and stably inherited 1. stably inherited: when a cell divides, the plasmid divides and makes two copies that goes into both daughter cells, thus stable in the pop 5 12. plasmids stably maintain themselves through common strategies 13. autonomous replication & copy number control 1. they autonomously replicate themselves independent of the host (may borrow DNAPs from host), have their own factors to drive replication 14. providing a useful function for the cell such as drug resistance 1. try to provide useful function, improve fitness of the host cell 15. encode partition systems that actively segregate the plasmid to daughter cells 1. makes sure each daughter cell inherits one 22 16. several types of plasmids 17. biologists take naturally occurring plasmids, modified the DNA for our own use ex. cloning 1. all plasmids used in the lab are derivatives of naturally occurring plasmids 18. range of diff sizes, diff copy numbers, wt usu have lower copy number range 6 19. two common ways of plasmid replication 20. origin of replication 1. ori vegetative (vegetative origin) 2. when bidirectional, replication forks may move differently 1. one replication fork may have diff properties than the other one 21. replication proteins 1. diff plasmids have diff requirements 2. generally use host-encoded polymerase w or w/o plasmid-encoded proteins 7 22. method of plasmid replication: rolling-circle replication 23. a plasmid takes one strand of itself and nicks it and copies itself such that the nicked strand gets displaced like toilet paper coming off the roll 24. there is a single stranded origin of replication and a double-stranded origin of replication 25. typically, a plasmid-encoded protein, Rep (relaxase) will nick the plasmid at a certain structure and covalently attach itself to that end of the strand it nicked 26. there is a free 3’ end where the Rep sits 27. now, DNAP can come along and copy the inner strand and indoing so is pushing off and displacing the outer strand while making an identical copy of the outer strand 28. keeps going around in a circle, pushing off what it previously copied into one big piece of ssDNA which once again gets nicked, and then repaired by a DNA ligase (gyrase) resulting in a ssDNA of the plasmid that gets filled in through the action of the double stranded origin of replication 9 29. method of plasmid replication: theta replication 30. can be unidirectional or bidirectional 31. there is a leading strand replication fork 32. end product of theta replication are concatemers (two circles that are linked) 33. needs an additional step where a topoisomerase that breaks both strands or one and passes the other strand it through and reseals the nicked plasmid - this process is called resolution, conferred by resolvases (type of topoisomerass) that are encoded by the plasmid itself 34. typically, those resolvases are encoded by the plasmid itself 10 35. plasmids appear in strains when you put them under selective pressure ex. antibiotic tx 36. you end up getting monster plasmids that encode multiple genes that confer antibiotic resistance (or resistance to other toxic materials) 37. this dramatically improves the recipient bacteria’s fitness 38. oriT & transfer genes (conjugation mechanisms) allows plasmid to jump from cell to cell – can even jump from gm- to gm+ 11 39. there is a big range of copy numbers 40. each plasmid has a unique copy number 41. F plasmid has 1 copy per E. coli chromosome – stringent plasmid 42. relaxed plasmid: more copies and doesn’t stringently control its own replication 12 How plasmids control copy number (through conc of protein/RNA) 43. the ColE1 replication mechanism 44. origin of replication 45. need to make a primer for DNA replication to start 46. involves 2 RNA molecules to make copies of itself 1. one also serves as a primer 2. the other antagonizes the action of that primer 47. the ratio of these two RNAs dictate whether the plasmid will make a copy of itself or stop 48. the more plasmids, the more inhibitory RNA it accumulates, that slows down the replication 49. when not much plasmid, there are more primer RNA 50. two RNAs antisense to one another: RNA I & RNA II 51. are transcribed in diff direction 52. concentration of RNA I i
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