Gene Regulation in Bacteria: Operons and Transcription Control

1. Ch 18 Gene Regulation

2. HOMEWORK • Tuesday • Read and outline 18.1 • We are not covering this in notes. Be thorough! • Make note of any questions you may have for when we go over this in class. • Answer concept check 18.1 • Due Wednesday 3-2 • Wednesday • Read and outline 18.3 • We are not covering this in notes. Be thorough! • Make note of any questions you may have for when we go over this in class. • Answer concept check 18.3 • Due Thursday 3-3

3. Thursday • Read and outline 19.2 and 19.5 • Concept check for these sections • Due Friday 3-4

4. REGULATING OFF DIGEST DOESN’T BUILD • Gene Regulation in Bacteria • Cells vary amount of specific enzymes by _______________ gene transcription • turn genes on or turn genes ______ • Turn genes on example: • if bacterium encounters new sugar (energy source), like lactose, then it needs to start making enzymes used to _______________ lactose • Turn genes off example: • if bacterium has enough tryptophan then it _______________ need to make enzymes used to _______________ tryptophan

5. FUNCTIONS RNA POLYMERASE TRANSCRIPTION SINGLE REPRESSOR • Bacteria group genes together • Operon • Genes grouped together with related _______________. • Example: all enzymes in a metabolic pathway • Promoter = __________________ binding site • Single promoter controls _______________of all genes in operon • Transcribed as one unit & a _________ mRNA is made • Operator = DNA binding site of _______________ protein

6. PROMOTER TRANSCRIBED MODEL OPERATOR • Operon Model • When gene is turnedON: • Polymerase binds to _______________ • Gene is _______________ • Operon: • Operator, promoter & genes they control: serve as a ______________ for gene regulation • When gene is turned off: • Repressor binds to ___________ site • Blocks RNA Polymerase • Diagram

8. REPRESSOR BLOCKING TRANSCRIPTION • How can genes be turned off? • _______________protein • binds to DNA at operator site • _______________RNA polymerase • blocks _______________

9. REPRESSIBLE ENZYMES NEEDED INACTIVE ENZYMES PROTEIN BIND REPRESSES ALLOSTERIC • Two types of Operons: • _______________ operons: can be turned off • Example: trpoperon: makes __________ used in tryptophan synthesis • Gene is on when tryptophan is _______________ • Repressor protein exists as an __________ form • Cell makes _________ for tryptophan synthesis • Synthesis pathway model • When excess tryptophan is present, it binds to trp repressor _______________ & triggers repressor to ______ to DNA • Blocks (__________) transcription • When tryptophan is present • need to make tryptophan-building enzymes • Tryptophan is _______________ regulator of repressor protein

10. trp operon 1trp operon 2 (Watch half only)trp operon 3

11. INDUCIBLE GLUCOSE OFF RELEASE • ___________ operons: can be turned on • Example: lacoperon: makes enzymes used in lactose digestion. • _______________is food of choice • Don’t need lactose digesting enzymes • Gene is turned _____ • Digestive pathway model • When lactose is present, binds to lac repressor protein & triggers repressor to _______________ DNA • Induces transcription • What happens when lactose is present? • Need to make lactose-digesting enzymes • Lactose is allosteric regulator of repressor protein

12. LINKS • choose narrated • Boring Man 1 • Boring Man 2 • lac operon phet

14. ANABOLIC SYNTHESIZING CATABOLIC AVAILABLE • Operon Summary • Repressible operon • usually functions in _______________pathways • _______________ end products • when end product is present in excess, cell allocates resources to other uses • Inducible operon • usually functions in _______________pathways, • digesting nutrients to simpler molecules • produce enzymes only when nutrient is _________ • cell avoids making proteins that have nothing to do, cell allocates resources to other uses

15. RAPIDLY EXTERNAL TRANSIENT REVERSABILITY ADJUST • Control of Eukaryotic Genes • Evolution of Gene Regulation • Prokaryotes • Single-celled • Evolved to grow & divide _______________ • Must respond quickly to changes in _________ environment • exploit _______________ resources • Gene regulation • turn genes on & off rapidly • flexibility & _________________ • _______ levels of enzymes for synthesis & digestion

16. INTERNAL HOMEOSTASIS GROWTH WHOLE • Eukaryotes • Multicellular • Evolved to maintain constant _______________ conditions while facing changing external conditions • __________________ • Regulate body as a whole • _______________& development • Specialization • Turn on & off large number of genes • Must coordinate the body as a _______________ rather than serve the needs of individual cells

17. PATHWAY CONTROL TRANSCRIPTION ENHANCER PROTEINS ENHANCED • Points of Control • The control of gene expression can occur at any step in the _______________ from gene to functional protein • Transcription • Control regions on DNA • Promoter • Nearby _______________ sequence on DNA • binding of RNA polymerase & _______________ factors • “base” rate of transcription • _________________ • distant control sequences on DNA • binding of activator _______________ • “_______________” rate (high level) of transcription

18. Pg 365 fig 19.5 • On a separate piece of paper, please compare/contrast the trp operon and the lac operon.

19. EXONS AMOUNT WEEKS INTERFERING BIND DEATH • mRNA processing • Alternative RNA splicing • variable processing of _______ creates a family of proteins • mRNA transport • Life span of mRNA determines ____________ of protein synthesis • mRNA can last from hours to _______________ • Small _______________RNAs (siRNA) • short segments of RNA (21-28 bases) • _____ to mRNA • create sections of double-stranded mRNA • “_______________” tag for mRNA • triggers degradation of mRNA

20. 5’ ATTACHMENT PROTEIN TRANSPORT UBIQUITIN PROTEASOME • Translation • Block initiation of translation stage • regulatory proteins attach to ____ end of mRNA • prevent _______________ of ribosomal subunits & initiator tRNA • block translation of mRNA to _______________ • Protein processing and Degradation • Protein processing • folding, cleaving, adding sugar groups, targeting for _______________ • Protein degradation • _______________tagging • _______________degradation

21. LABEL POLYPEPTIDE RAPIDLY MACHINE DISPOSER PROTEINS RECYCLING • Ubiquitin • “Death tag” • mark unwanted proteins with a _______________ • 76 amino acid _______________, ubiquitin • labeled proteins are broken down __________ in "waste disposers" • proteasomes • Protein-degrading “_______________” • cell’s waste _______________ • breaks down any _______________into 7-9 amino acid fragments • cellular _______________

22. Pg 370 fig 19.10 • On the same sheet of paper from yesterday, please describe 4 different ways eukaryotic genes are controlled.

23. Chapter 20 DNA Technology and Genomics

24. SPECIES DIFFERENT ENGINEERING PRACTICAL HORMONES (HGH) ORGANISMS PRODUCTS SELECTIVE • Understanding and Manipulating Genomes • Recombinant DNA (do not confuse with recombinants in genetics) • Definition- DNA in which nucleotide sequences from two __________________ sources (__________) are combined in vitro into the same DNA molecule. • Basis of genetic __________________ • Def: manipulation of genes for ______________ purposes • Examples: Protein products such as __________________ and blood-clotting factors, much more. • Biotechnology- manipulation of ______________ or their components to make useful __________________. • Ex: microbes used to make wine and cheese, ___________ breeding (exploiting naturally occurring mutations of genetic recombination.)

25. LONG NON-CODING 100,000 CLONING MULTIPLE SIZED • DNA Cloning • Problems with DNA • DNA molecules are very ________. • Genes occupy only a small portion of DNA (others are __________________ nucleotides) • Gene may be only 1/__________________ of a chromosome. • Gene __________________- method to prepare well-defined gene-________ pieces of DNA in __________________copies.

26. PLASMIDS REPLICATE ISOLATED INSERTED RETURNED RECOMBINANT CLONE MULTIPLE • Cloning and Applications • Common approach of DNA cloning uses bacteria (E. coli) and their __________________. • Plasmids- small circular DNA molecules that _____________ separately from bacterial chromosomes • Plasmids are first __________________ from bacterial cells • Foreign DNA is then ______________ into the plasmid. • Resulting plasmid is __________________ to bacterial cell producing a __________________bacterium • It can then reproduce to form a ____________. • Clone of cells contains __________________copies of the gene.

28. GENE PROTEIN RESEARCH METABOLIC RESISTANCE PROTEINS CULTURES RARE AVAILABLE • Cloned genes are useful: • To make many copies of a particular _________. • To produce a __________________ product • Copies of cloned genes can be isolated for __________________ or to give an organism a new __________________capability. • Ex. Pest __________________ can be transferred into plants of different species. (Potato famine) • __________________ such as human growth hormone can be harvested from bacterial ___________ carrying the cloned gene for a protein. • Why is DNA cloning valuable • Protein-coding genes are very ________- 1 ppm, so the ability to clone such rare fragments can make something rare more ___________________.

29. ENDONUCLEASES CUTTING • Restriction Enzymes • Definition- restriction _______________ protect the bacterial cell against intruding DNA and work by ________ foreign DNA (restriction).

30. SPECIFIC POINTS • How they work: • Restriction enzymes are ________________ and recognize a particular DNA sequence (restriction site). • They then cut both DNA strands at specific ________ within the restriction site. • DNA of bacterial cell is __________________ from cell’s own restriction enzymes by adding _______________ (_____) groups to adenines or cytosines within sequences recognized by enzyme. PROTECTED METHYL CH3

32. SYMMETRICAL NUCLEOTIDES FRAGMENTS SAME CLEAVE STAGGERED STICKY END • More details about restriction enzymes • Most sites are __________________: nucleotides are identical when read from 5’3’ direction. • Ex: 5’GAATTC 3’ • Most recognize sequences of 4-8 __________________. • Restriction __________________- pieces of DNA cut by restriction enzymes. • Same restriction enzymes result in _______________ fragments. • Useful restriction enzymes ___________ sugar-phosphate backbones in both DNA strands in a ______________ way. • Resulting double stranded fragments have at least one single stranded end- __________________

33. BASE PAIRS Restriction enzyme animation OTHER LIGASE • Sticky ends can form hydrogen-bonded __________________ with complementary sticky ends on ___________ DNA molecules cut with the same enzyme. • DNA ____________ can permanently bind the two fragments by forming a covalent bond between the sugar-phosphate backbone.

35. VECTOR REPLICATES • Cloning Eukaryotic Gene in Bacterial Plasmid • Original plasmid is a cloning __________________- DNA molecule can carry foreign DNA into a cell and __________________ there. • Cloning vectors can be isolated, manipulated to form __________________ plasmids by insertion of foreign DNA in vitro, and then __________________into bacterial cells. • Cells reproduce rapidly and __________________ foreign DNA carried. • plasmid vector RECOMBINANT REINTRODUCED MULTIPLY

36. AMPICILLIN LACTOSE DIGESTED MANY MIXED COMPLEMENTARY JOINING • Producing Clones of Cells not boring manboring man • Human genomic DNA is isolated and vector from bacterial plasmid is isolated. • Plasmid carries two genes: ampR (E.coli resistant to __________________) and lacZ (encodes B-galactosidase which hydrolyzes __________________). lacZ gene contains a single copy of restriction site. • Both plasmid and human DNA are ___________ by the same restriction enzyme producing sticky ends. Plasmid is cut once while human DNA is cut at __________________ fragments. • The human DNA and plasmid fragments are __________________ which allows base pairing between __________________ sticky ends. DNA ligase is then added __________________ plasmid and human DNA fragments.

37. HUMAN NONRECOMBINANT MUTATION HYDROLYZE TRANSFORMATION ANOTHER AGAR TRANSFORMED • Resulting plasmids can be recombinant (contain __________________ DNA), be a combination of two plasmids, or a ____________________version of the original plasmid. • DNA resulting from step c is then mixed with bacteria with a __________________ in lacZ gene making them unable to __________________ lactose. • Cells take up foreign DNA by __________________. • Some cells acquire a recombinant plasmid with gene of interest and others acquire plasmid with _________ gene. • Cloning step: bacteria are plated on ______________(nutrient medium) containing ampicillin and X-gal. • This allows us to identify clones of cells __________________with recombinant plasmid.

39. RESISTANCE DIVIDE COLONY COLOR BLUE BLUE FUNCTIONAL • How to recognize clones carrying recombinant plasmids • Only cells with plasmid will reproduce since they have ampR gene allowing __________________to ampicillin in the medium and will __________________. • Once clone contains 105 cells it can be seen as a _________________ on the agar. • __________________of colonies allows us to determine colonies of bacteria with recombinant plasmids from those with nonrecombinant plasmids. • Colonies with nonrecombinant plasmids with intact lacZ are __________________ because they produce B-galactosidase which hydrolyzes X-gal and produces a __________________ product. • Colonies with recombinant plasmids will be white because there is no __________________B-galactosidase.

40. Each dot represents a colony of cells.

41. HYBRIDIZATION ANOTHER PROBE SINGLE STRANDED • Identifying Clones Carrying Gene of Interest • Look for the Gene Itself • Nucleic Acid __________________- DNA of the gene is detected by ability to base-pair with complementary sequence on __________________ nucleic acid molecule. • If a part of the nucleotide sequence of the gene of interest is known, a nucleic acid ______________ can be used. • Nucleic acid probe- a short complementary molecule of __________________________ nucleic acid (DNA or RNA). • Example: sequence of DNA on gene of interest: 5’ GGCTAACT 3’

42. Take out a sheet of paper and, using your notes and book, write a brief outline of how genes are cloned. • Suggested words to include: plasmid, vector, bacteria, restriction enzymes, ligase. • (HINT: pretend you are explaining the “Circle and Bacon” picture to a younger friend/relative/person.)

43. RADIOACTIVE DESIRED DENATURE • Probe molecules are labeled with __________________ isotopes so that it can be tracked. • The probe molecules will hydrogen bond to the complementary part of the __________________ gene. • Heat or chemicals are used to __________________the cells’ DNA to make it single stranded and able to base pair with the probe.

44. TRANSFERRING DENATURE INCUBATED RADIOACTIVE LOCATED • Example • Filter paper is pressed against the plate __________________ cells to the filter. • The filter is treated to break cells and __________________ DNA. • Radioactive probe molecules are __________________ with the filter and allows it to base pair with complementary DNA. • Filter paper is laid under film while exposing __________________ areas. • Colonies carrying gene of interest have been _________.

45. SEGMENTS LIBRARY LARGER cDNA PART TRANSCRIBED TRANSCRIPTASE DNA mRNA • Cloned Genes in DNA Libraries • Genomic _________- set of thousands of DNA ____________ from a genome, each carried by a plasmid, phage, or other vector. • Advantage of phages over plasmids is that phages can carry a _________ DNA segment. • Complementary DNA(___________) Library • Only represents ____________ of a genome- genes that were ____________ into mRNA. • Start with mRNA extracted from cells, reverse __________________ (RT) is used to make single stranded _______ from _______, then DNA polymerase will complete the second strand of DNA.

47. TYPE UNABLE REGULATORY CODING FUNCTIONS PATTERNS EXPRESSION • Use of DNA libraries • Genomic library use • Want to clone a gene but unsure in what cell ______ it is expressed or _________ to obtain cell type. • Interested in _______________ sequences or introns of a gene. • cDNA use • interested in _______________ sequence of genes • Studying genes responsible for specialized _______________ of cell types • Changes in _______________ of gene _______________

48. DIFFERENCES PROKARYOTIC READING CONTINUES SYNTHESIS • Cloning and Expressing Eukaryotic Genes • Having cloned eukaryotic gene function in a bacterial host can be difficult due to the _______________ of gene expression in prokaryotes and eukaryotes. • Expression vector- cloning vector that contains a highly active _______________ promoter upstream of a restriction site where eukaryotic gene can be inserted in the correct _______________ frame. • Bacterial host recognizes the promoter and ___________ to express the foreign gene. • Expression vectors allow _______________of eukaryotic proteins in bacterial cells.

49. INTRONS cDNA YACs PLASMIDS COMBINE NORMALLY LONGER PLASMA MEMBRANES • Another problem can result when _______________are present in eukaryotic genes and can be solved by using ______ (only exons). • Yeast Artificial Chromosomes (_________) • Yeast cells are single-celled fungi that are easy to grow and have _______________. • YACs _______________eukaryotic chromosome with foreign DNA. • YACs act and divide ___________ in mitosis and can carry _______________ DNA segment than plasmid vectors. • Electroporation • Brief electrical pulse applied to a solution containing cells creating brief holes in their ________________________ allowing DNA to enter.