All living things have a genetic molecule

1. All living things have a genetic molecule • In prokaryotes and eukaryotes: DNA • Even in viruses, genetic material is DNA or RNA • Directs day to day operations of the cell • Provides instructions for making a new individual • passed on to daughter cells during cell division • Eubacteria and Archaea differ in genome structure • Focus is on Eubacteria

2. Chromosome Organization • Most bacteria genomes are single, covalently closed, circular DNA molecule • Others may have a linear molecule or several pieces • DNA is negatively supercoiled • DNA is slightly underwound • Underwinding carried out by DNA gyrases • Makes separation of strands easier during transcription • Supercoiling creates twisted loops • A section of supercoiled DNA is a domain • About 50 domains estimated to exist

3. Packaging of E. coli DNA DNA is “packaged” with proteins. Together, this is called the nucleoid. Note arrows: one shows where the DNA has been “nicked”, relaxing the supercoiling. The other points to a supercoiled region. That supercoiling can be relaxed in ONE PLACE means that the DNA is constrained in places.

4. What’s in the DNA • Genes. Lots of genes. • A gene is a section of DNA with the information for making a protein (or an RNA) • DNA codes for rRNAs and tRNAs as well • Prokaryotic DNA differs from eukaryotic • Many eukaryotes have non-coding DNA = junk • Up to 90% or more of DNA is junk in eukaryotes • Relatively little spacer (non-coding) DNA between genes in prokaryotes • Some Archaea have introns, but none in Eubacteria.

5. Bacteria have transposons • A bacterial genome has a dozen or so • “jumping genes”, pieces of DNA that copy themselves • DNA either cuts out, inserts elsewhere or • Copies itself and copy inserts elsewhere • Simple: Insertion sequences; • Code for transposase and repressor • Composite transposons • Insertion sequences which flank other DNA • Typically antibiotic resistance genes

6. Plasmids • Plasmids: small, circular, independently replicating pieces of DNA with useful, not essential info. • 1% to 10% of genome • Types of plasmids • Fertility, • resistance, • catabolic, • bacteriocin, • virulence, • tumor-inducing, and • cryptic http://www.estrellamountain.edu/faculty/farabee/biobk/14_1.jpg

7. About plasmids-1 Fertility plasmid: genes to make a sex pilus; replicates, and a copy is passed to another cell. Resistance plasmid: genes that make the cell resistant to antibiotics, heavy metals. Catabolic plasmid: example, tol plasmid with genes for breaking down and using toluene, an organic solvent. www.science.siu.edu/.../ micr302/transfer.html

8. About plasmids-2 • Bacteriocin plasmid: codes for bacteriocins, proteins that kill related bacteria. • Virulence plasmid: has genes needed for the bacterium to infect the host. • Tumor-inducing plasmid: The Ti plasmid found in Agrobacterium tumefaciens. Codes for plant growth hormones. When the bacterium infects the plant cell, the plasmid is passed to the plant cell and the genes are expressed, causing local overgrowth of plant tissue = gall. Very useful plasmid for cloning genes into plants. • Cryptic: who knows?

9. Gene structure Some sequences mark the beginning of the information, providing binding sites for proteins.This is followed by the information for making the proteins. A termination sequence signals that making mRNA should end.

10. The Genetic Code-2 http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif

11. Transcription: making mRNA • RNA a polymer assembled from monomers • Ribonucleoside triphosphates: ATP, UTP, GTP,CTP • RNA polymerase • Multi-component enzyme • Needs a template, but NOT a primer • In bacteria, a component (sigma) recognizes the promoter as the place on DNA to start synthesis • Synthesis proceeds 5’ to 3’, just as in DNA • mRNA is complementary and antiparallel to the DNA strand being copied.

12. Transcription-2 • The order of nucleotides in the RNA reflects the order in the DNA • If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand. Either DNA strand may contain the gene! Transcription just runs the other direction.

13. Sense, antisense Compare the sense strand of the DNA to the mRNA. Note that mRNA synthesis will be 5’ to 3’ and antiparallel. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif

14. Sigma subunit recognizes promoter region of DNA http://cats.med.uvm.edu/cats_teachingmod/microbiology/courses/gene_regulation/images/dij.tc.elong1.jpg

15. The Process of Transcription-2 • RNA synthesis continues (Elongation), only one DNA strand (template) is transcribed. • RNA nucleotides, complementary to bases on DNA strand, are connected to make mRNA • Termination: must be a stop sign, right? • In bacteria, hairpin loop followed by run of U’s in the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next. Most common. OR • Termination factor “rho”. Enzyme.Forces RNA polymerase off the DNA.

16. Termination of Transcription in Bacteria The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart. http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif

17. Ribosome schematic http://staff.jccc.net/pdecell/proteinsynthesis/translation/elongation12.gif

18. Translation-3 • Termination • When stop codon is in A site, no tRNA binds • GTP-dependent release factor (protein) removes polypeptide from tRNA in P site. All done. • Ribosomal subunits typically dissociate. • Do a Google Search for translation animation • Many hits. Note presence, absence of E site • Note shape of ribosomes • Note whether role of rRNA in catalysis is shown

19. Polysomes Multiple ribosomes attach to the mRNA and begin translating. Strings of ribosomes can be seen attached to the mRNA. http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-29b-bottom.GIF www.cu.lu/labext/rcms/ cppe/traducti/tpoly.html

20. Simultaneous transcription and translation • No processing, no nucleus; mRNA already where the ribosomes are, so they get started quickly. http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-30.GIF

21. Using DNA is expensive to the cell • Model: protein of 50,000 daltons • Approximately 500 amino acids, so 500 codons • 500 codons is 1500 RNA bases • Transcription • Every base added (NTP to NMP) spends 2 phosphates, so equivalent of 2 ATP • 1500 bases/mRNA = 3000 ATP • Translation • 1 ATP to move ribosome, 1 to charge tRNA /codon • 2 ATP/ codon = 2 * 500 = 1000 ATP for every single protein molecule produced.

22. Bacteria tightly regulate their activities Bacteria must respond quickly to changes in the environment. Bacteria are small compared to their environment, have no real capacity for energy storage. Simultaneous transcription and translation allows them to synthesize the proteins they need quickly. Wasteful activities are avoided. If there are sufficient amounts of some metabolite, bacteria will avoid making more AND avoid making the enzymes that make the metabolite.

23. Feedback inhibition of pathways

24. More on Regulation • In biochemical regulation, processes like feedback inhibition prevent wasteful synthesis. • To save more energy, bacteria prevent the synthesis of unneeded enzymes by preventing transcription. • In operons, several genes that are physically adjacent are regulated together. • Two important patterns of regulation: Induction and repression. • In induction, the genes are off until they are needed. • In repression, the genes normally in use are shut off when no longer needed.

25. Operons and Regulons • Nearly 50 years ago, Jacob and Monod proposed the operon model. • Many genes in prokaryotes are grouped together in the DNA and are regulated as a unit. Genes are usually for enzymes that function together in the same pathway. • At the upstream end are sections of DNA that do not code, but rather are binding sites for proteins involved in regulation (turning genes on and off). • The Promoter is the site on DNA recognized by RNA polymerase as place to begin transcription. • Operator is location where regulatory proteins bind. • Promoter and Operator are defined by function.

26. Binding of small molecules to proteins causes them to change shape Characteristic of many DNA-binding proteins When the small molecule binds to the protein: Inducible operons: Repressor protein comes off DNARepressible operons: Repressor protein attaches to DNA

27. Binding of lac repressor to DNA More lac Repressor Research - Lewis & Lu Labs

28. Genetic regulation • Genotype is not phenotype: bacteria possess many genes that they are not using at any particular time. • Transcription and translation are expensive; why spend ATP to make an enzyme you don’t need? • Operon • Genes physically adjacent regulated together • Regulon • Genes dispersed but controlled by same proteins • Operator sequences must be same/similar

29. More on Regulation • Two important patterns of regulation: Induction and repression. • In induction, the genes are off until they are needed. • In repression, the genes normally in use are shut off when no longer needed. • Negative control • Binding of protein to the DNA prevents transcription • Positive control • Binding of protein to DNA promotes transcription

30. Repressible operons • Operon codes for enzymes that make a needed amino acid (for example); genes are “on”. • Repressor protein is NOT attached to DNA • Transcription of genes for enzymes needed to make amino acid is occurring. • The change: amino acid is now available in the culture medium. Enzymes normally needed for making it are no longer needed. • Amino acid, now abundant in cell, binds to repressor protein which changes shape, causing it to BIND to operator region of DNA. Transcription is stopped. • This is also Negative regulation (protein + DNA = off).

31. Repression picture Transcription by RNA polymerase prevented.

32. Regulation can be fine tuned The more of the amino acid present in the cell, the more repressor-amino acid complex is formed; the more likely that transcription will be prevented.

33. Structure of the Lac operon KEY: P O are the promoter and operator regions. lac Z is the gene for beta-galactosidase. lac Y is the gene for the permease. lac A is the gene for a transacetylase. lac I, on a different part of the DNA, codes for the lac repressor, the protein which can bind to the operator.

34. How the lac operon works When lactose is NOT present, the cell does not need the enzymes. The lac repressor, a protein coded for by the lac I gene, binds to the DNA at the operator, preventing transcription. When lactose is present, and the enzymes for using it are needed, lactose binds to the repressor protein, causing it to change shape and come off the operator, allowing RNA polymerase to find the promoter and transcribe. http://www.med.sc.edu:85/mayer/genreg1.jpg

35. Lactose is not actually the inducer Low basal levels of beta-galactosidase exist in the cell. This converts some lactose to the related allolactose which binds to the lac repressor protein. Synthetic inducers such as IPTG with a similar structure can take the place of lactose/allolactose for research purposes. http://www.search.com/reference/Lac_operon

36. Glucose is the preferred carbon source

37. Positive regulation • Presence of lactose is not enough • In diauxic growth graph, lactose is present from the start. Why isn’t operon induced? • Presence of glucose prevents positive regulation • NOT the same as inhibiting • Active Cyclic AMP receptor protein (CRP) needed to bind to DNA to turn ON lactose operon (and others) • Presence of glucose (preferred carbon source) prevents activation of CRP. www.answers.com/.../catabolite-activator-protein

38. Global control: modulons • Different operons/regulons affected by same environmental signal • Presence of glucose • Change from O2 to anaerobic growth • Nitrogen limitation; phosphate starvation • Growth rate control • Cell division • Stationary phase; entering starvation state • One method of control: alternate sigma factors • Sigma controls which promoters are used