Vocabulary Operon Promoter Terminator Enhancer Regulatory Gene Inducer

Vocabulary… Operon Promoter Terminator Enhancer Regulatory Gene Inducer Repressor Regulatory Protein/Sequence ◦ Inducible and repressible Positive gene control Negative gene control Lac Operon Trp Operon Plasmid Transformation Conjugation Retrovirus Lytic cycle Lysogenic cycle

operator: “switch” that controls access of RNA polymerase to genes for transcription repressor: a protein that binds to the operator to block transcription ◦ corepressor: small molecule that binds to repressor to make it active (helps block transcription) regulatory gene: gene that code for the production of repressors (always on at a low rate) Regulatory sequence: stretch of DNA that interacts with regulatory proteins to control transcription (promoter, terminator, enhancer) inducer: small molecule that inactivates the repressor (allows transcription) 2

Gene Expression Biotechnology Bacteria and Viruses Chapters 18, 19, and 20 3

Genes regulated at the transcription stage (mostly). ◦ feedback inhibition (positive and negative) Important in embryonic development (eukaryotes) to create differentiated cells Also important in cancer prevention and regulation of the cell cycle There are differences in the way prokaryotes and eukaryotes regulate gene expression…why? ?

Prokaryotic Gene Regulation Express only those genes that are needed by the cell (allows for efficient metabolism) ◦ Adjust activity of enzymes ◦ Adjust production level of enzymes Genes switched on/off as needed due to environmental conditions ◦ Operon model: basic mechanism for the control of gene expression in bacteria

Operons… Operon: entire stretch of DNA required to produce a protein (enzyme)—under the control of a single promoter ◦ this may include several genes operator + promoter + genes Negative Gene Regulation (operon switched off by repressor) ◦ Lac operon (inducible) ◦ Trp operon (repressible)

Lac Operon inducible operon. usually “off” but can be turned on by allolactose (sugar--isomer of lactose) ◦ inactivates repressor to allow for transcription genes code for 3 enzymes that utilize lactose Figures 18. 4(a) and 18. 4(b) 7

Trp Operon repressible operon. controls synthesis of tryptophan (amino acid) is usually “on”, but can be switched off by a repressor. (prevents transcription) ◦ controlled by a regulatory gene (trp. R). (always expressed at a low rate) allosteric regulation! Increased concentration of tryptophan will result in less of it synthesized by bacteria Figures 18. 3(a) and 18. 3(b) 8

Positive gene regulation (regulatory protein interacts directly with genome to switch transcription on) Positive feedback! ◦ Cyclic AMP (c. AMP) and Lac operon bacteria preferentially use glucose for energy (glycolysis) but will use lactose in its absence (lac operon switches on) c. AMP accumulates when glucose is scarce enhances the production of enzymes from the lac operon (directly stimulates gene expression) Binds to specific region in promoter also has the ability to enhance the transcription of other genes (not just lac)

Bacteria Three shapes (cocci, bacilli, spirilla) Gram+ and Gram◦ Cell wall type lack complex compartmentalization (prokaryotes) ◦ circular chromosome located in nucleoid (region of cytoplasm)

◦ Plasmids (independently replicating DNA carrying few genes) R plasmid (carries resistance genes for specific antibiotics) -MRSA (methicillin resitance) -Multi-drug resistant strains -Enterococcus sp. -Pseudomonas sp. -Tuberculosis Implications for human and veterinary medicine, as well as agriculture 11

Bacterial Reproduction No sexual reproduction ◦ asexual (binary fission) New mutations increase genetic diversity rapidly due to short generation times (rapid evolution) 12

Bacterial Reproduction Horizontal Gene Transfer… ◦ Transformation (take in foreign DNA from its surroundings) produces recombinant cells (DNA from two different cells) ◦ Transduction (bacteriophages--viruses carry genes from one cell to another) recombinant cells produced ◦ Conjugation (DNA transferred between two cells--one cell donates the other receives) ◦ Transposition (DNA segments move with and between molecules) “jumping genes”—move from chromosome to plasmid 13

Eukaryotic Gene Regulation No Operons!! Differential gene expression: different genes expressed by cells within the same genome ◦ Depends on cell function or stage of development Like prokaryotes, most often regulated at the stage of transcription (but much more complex) ◦ Eukaryotes do have the ability to control gene expression at every step of the process

How it works… http: //www. bozemanscienc e. com/031 -gene-regulation/ Transcription factors need to be present for the process to begin ◦ These bind to specific DNA sequences “upstream” of the gene to be expressed (regulatory regions— enhancer/promoter) Attracts RNA polymerase to attach ◦ Controlling availability of these factors in a cell is a major component in regulating gene expression Some factors are activators (increase expression) others are repressors (decrease expression) ◦ These determine what (or if) genes will be expressed (and how much) This changes phenotype of the organism! 15

Applications to Biotech Plasmids can be used to genetically engineer organisms, also to further study specific genes ◦ Easily moved between organisms, easily manipulated restriction enzymes (restriction endonucleases) are used to cut DNA at specific sequences (restriction sites) ◦ Produces blunt or sticky ends (depends on cut) ◦ Hundreds have been identified, each recognizes a specific site Eco. R 1 cuts at GAATTC 16

DNA (cut with the same enzyme) is then combined with other DNA making a recombinant DNA sequence ◦ Base-pairing occurs, ligase seals break ◦ This can produce some very interesting organisms! It allows for cells to produce proteins they wouldn’t normally. Insulin, vaccines, oil spill clean up, agriculture, etc. 17