Bio 101A GE III results day

Which one of the following occurs when RNA polymerase attaches to the promoter DNA? A) elongation of the growing RNA molecule B) termination of the RNA molecule C) addition of nucleotides to the DNA template D) initiation of a new RNA molecule E) initiation of a new polypeptide chain

Which of the following is the enzyme that HIV uses to synthesize DNA on an RNA template? A) ligase B) RNA polymerase C) terminator enzyme D) reverse transcriptase E) None of the choices are correct.

Prokaryotic Gene Regulation Bio 101A Operon structure and function Positive/Negative gene regulation Promoters, operators, repressors, inducers

Q: What is “regulation”? "When I was warning about the danger ahead on Wall Street months ago because of the lack of oversight, Senator McCain was telling the Wall Street Journal -- and I quote -- 'I'm always for less regulation.' " – Sen. Barack Obama “Senator Obama was silent on the regulation of Fannie Mae and Freddie Mac, and his Democratic allies in Congress opposed every effort to rein them in…last year he said that subprime loans had been, quote, “a good idea.””- Sen. John McCain

Enzymes are coded for by genes DNA is the code to make proteins Enzymes are made of protein In order for a cell to make an enzyme, it must access the DNA for that enzyme Enzymes are very specific to their task

V. fischeri interacts symbiotically with a squid Helps the squid camouflage itself during nocturnal hunting 95% of colonies are expelled daily The rest are fed in pouches in the squid’s tissue Bacterium has an interest in regulating expression of luciferase gene

V. fischeri interacts symbiotically with a squid The winnowing: establishing the squid–vibrio symbiosis Spencer V. Nyholm & Margaret McFall-Ngai Nature Reviews Microbiology 2, 632-642 (August 2004)

Enzyme Regulation maintains Efficiency • Enzymes: General Background • b - galactosidase • Gene Regulation • Biochemical Regulation • Experiment 9

b - galactosidase β-galactosidase is our enzyme of choice. This enzymatic protein is a tetramer. That means that it is comprised of how many identical subunits? The active sites of this enzyme are located at the junctions of the subunits…here…here…here…and here. This enzyme is produced by many different organisms and has been very thoroughly studied. You will be studying its production in a bacteria and its activity using enzyme purified from a mold. Transition: So what is exactly is beta-galactosidase and what does it do?… 10

b - galactosidase galactose lactose b - galactosidase glucose H O 2 galactose Well, Humans also produce β-galactosidase though the human version of this enzyme is called lactase. Does anyone know what lactase deficiency is called? [No? ok./You’re right] Let me tell you how this enzyme works: β-galactosidase catalyzes the breakdown of a disaccharide, lactose, into two monosaccharides: glucose and galactose. Glucose is the prime energy source for many organisms. [Any ideas now?] In lactose intolerance, undigested lactose causes intestinal discomfort. Transition: We know that both the amount produced and the efficiency of individual β-galactosidase are tightly regulated, which begs the question… lactose b - galactosidase glucose (aka lactase in humans) 11

b - galactosidase Regulation • Why Regulate b - galactosidase ? • Levels at which b - galactosidase can be regulated: • Genetic • Biochemical Why do you think this enzyme is regulated? {-limited energy –production takes energy –environments vary –don’t waste energy}. I mentioned that both the amount and efficiency of the enzyme are regulated. Which sort of regulation do you think will happen at the genetic level? Which sort do you think will happen at the biochemical level, i.e., after the enzyme is produced? Transition: In this lab, your task will be to identify an unknown substance by examining how it regulates β-galactosidase at both the genetic and biochemical levels Caveat- as Albert pointed out, this is not regulated the same way in people as it is in E.coli. 12

The lac operon regulates when β-galactosidase is made

When lactose is present, transcription is activated

LE 18-20 Regulation of enzyme activity Regulation of enzyme production Precursor Feedback inhibition Enzyme 1 Gene 1 Enzyme 2 Gene 2 Regulation of gene expression Enzyme 3 Gene 3 Enzyme 4 Gene 4 Enzyme 5 Gene 5 Tryptophan

Operons: The Basic Concept An operon is a collection of prokaryotic genes transcribed together on a single mRNA transcript to serve a single purpose Composed of An operator, an “on-off” switch A promoter Genes for metabolic enzymes Can be switched off by a repressor protein A corepressor is a small molecule that binds to a repressor to switch an operon off

Polypeptides that make up enzymes for tryptophan synthesis Prokaryotic Operon structure ensures efficient regulation of transcription trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene RNA polymerase Start codon Stop codon 3¢ mRNA 5¢ mRNA 5¢ E D C B A Protein Inactive repressor Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on The tryptophan biosynthesis operon is repressible by the presence of its product, tryptophan

LE 18-21b_1 DNA mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off

LE 18-21b_2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present, repressor active, operon off

Basic Operon Regulation Repressor Protein RNA Polymerase NO TRANSCRIPTION Promoter Operator Structural Genes Repressor mRNA Regulator Gene

Tryptophan Operon Tryptophan Present NO TRANSCRIPTION trpR mRNA Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase NO TRANSCRIPTION trpR mRNA Q: Why might the cell want to produce an aporepressor that is only activated by the operon’s end product? + tryptophan (corepressor) TrpR protein (homodimer) TrpR aporepressor + corepressor (can bind to operator)

TrpR protein (homodimer) (cannot bind to operator) Tryptophan Operon Tryptophan Absent Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase TRANSCRIPTION trpR mRNA TrpR protein (homodimer) TrpR aporepressor (cannot bind to operator)

Tryptophan Repressor Protein TrpR protein subunits Tryptophan (co-repressor) DNA

Repressible and Inducible Operons: Two Types of Negative Gene Regulation A repressible operon is one that is usually on; binding of a repressor shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The classic example of an inducible operon is the lac operon

LE 18-22a Regulatory gene Promoter Operator DNA lacl lacZ No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off

LE 18-22b lac operon DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA 5¢ Permease Transacetylase Protein -Galactosidase Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on

Inducible enzymes usually function in catabolic pathways Repressible enzymes usually function in anabolic pathways Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP When glucose levels increase, CAP detaches from the lac operon, turning it off

Lactose present, glucose scarce (cAMP level high): abundant lac LE 18-23a Promoter DNA lacl lacZ RNA polymerase can bind and transcribe CAP-binding site Operator Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

Lactose present, glucose present (cAMP level low): little lac LE 18-23b Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

In a prokaryote, a group of genes with related functions, along with their associated control sequences, defines   A) an allele.   B) an operon.   C) a locus.   D) a transposon.   E) a chromosome.  

Operons can be cut and pasted together to make operon fusions Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal mutant trpR-containing plasmid

If the repressor is knocked out, what will happen in the presence of Tryptophan? Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal

What if we add a plasmid which contains the TrpR gene. With tryptophan What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? Tryptophan Operon Lactose Operon Repressor Pro. Oper. Att. TrpE, D, C, B, A lacI T Pro. Oper. Z gene Y gene A gene Promoter Operator Z gene Y gene A gene mRNA β-gal mutant trpR-containing plasmid

Another engineered plasmid with fusion Operon: pGLO HELWIG Another engineered plasmid with fusion Operon: pGLO Manufactured by a private corporation AraC- arabinose gene GFP- Green Fluorescent protein bla- Beta-lactamase ori- you know this… pGLO ori bla GFP araC 4/16/2017 VandePol

Is this: Anabolic or Catabolic. Positive or negative Is this: Anabolic or Catabolic? Positive or negative? Inducible or repressible? 4/16/2017

Expression of Green Fluorescent Protein How do you think this fusion was made? What are the structural sequences? The regulatory sequences? What happens when we add arabinose sugar to these bacteria? What do you think is meant by “reporter gene”?

lac Operon and ara operon Z Y A LacI Effector (Lactose) lac Operon B A D araC RNA Polymerase Effector (Arabinose) ara Operon RNA Polymerase 4/16/2017

Which colonies will glow? Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp LB/Amp/Ara LB 4/16/2017

Appendix: pGLO slides that may be helpful Stuff about GFP, arabinose, beta-lactamase, etc.

DNA polymerase binds to the ori LE 16-12 Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand Bubble Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene Effector (Arabinose) B A D araC RNA Polymerase Effector (Arabinose) ara Operon What will happen on the Ara (+) plates? What will happen on the Ara (-) plates?

Green Fluorescent Protein 40 Å 30 Å discovered in 1960s by Dr. Frank Johnson and colleagues closely related to jellyfish aequorin absorption max = 470nm emission max = 508nm 238 amino acids, 27kDa “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore amino acid substitutions result in several variants, including YFP, BFP, and CFP Previous Slide: Immunofluorescence Key Points of Current Slide: try to know the key points on this slide well so that you’re not reading it to the class. the absorption maximum is very close to that of the FITC fluorochrome, so in our lab we will be using the FITC filter to excite GFP CAs: FYI autofluorescence (when cells naturally fluoresce without the addition of a fluorochrome) occurs in the C. elegans gut; using a normal GFP filter this looks yellow, but under FITC filter looks green. Next Slide: GFP Fusion and Protein Localization Transition: GFP applications are slightly different from those of fluorophores that are just attached post-translationally to proteins or antibodies. Instead, the GFP gene is commonly fused to a given gene of interest.

Using GFP as a biological tracer GFP can be fused to cellular proteins Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

GFP Fusion & Protein Localization 1 Gene Fusion Gene X (no stop codon) Control Region GFP gene (in-frame with Gene X) express gene-of-interest 2 Translated Fusion Protein 3 Fluorescence Visualization Previous Slide: GFP Background Information Key Points of Current Slide: 1. GFP is ordinarily fused to the end of the gene encoding the protein of interest. The GFP gene has to be in-frame with the protein, which cannot have a stop codon. 2. Usually researchers create this fusion in such a way that the GFP::Protein X fusion is translated as one protein. Key is that GFP cannot alter Protein X folding, and more importantly, function. GFP will glow when excited. 3. Since GFP is part of fusion protein, it will localize wherever Protein X localizes. In this example, localizes to the inner mitochondrial matrix. Useful to test for (a) protein expression/repression and (b) distribution/localization of proteins within cells or organisms. Next Slide: Nucleic acid stains Transition: Finally, there are markers that are specific to nucleic acids GFP Protein X

Fusion protein in C. elegans highlights nervous system Vesicle Transport in the Caenorhabditis elegans Nervous System [START WEEK 1 TALK] Again, this is a title slide that you need not spend too much time on. You may want to excite the class by telling them that we took this picture in training, and that they will see very similar images during Week 2. Next Slide: System Objectives review Transition: Just to refresh your memory, we’ll begin with the system objectives again.

SNB-1::GFP Fusion 1 Fusion of snb-1 to GFP gene 2 Control Region For snb-1 GFP gene (in-frame with snb-1) express gene-of-interest 2 Neurotransmitter-Packaging Vesicles Present GFP-Tagged SNB-1 on Surface 3 View SNB-1 (Vesicle) Distribution Previous Slide: Experimental Outline Key Points of Current Slide: Fuse snb-1 gene (for synaptobrevin) to GFP gene Co-expression leads to translation of fusion protein SNB-1::GFP will be presented on the surface of neurotransmitter-packaging vesicles. GFP will glow wherever SNB-1 is localized in the worm (in sample picture, can see glowing nerve ring, which has a large collection of axons and dendrites) Blue = DAPI (cell localization) Next Slide: Transgenic worms Transition: We used a process called transformation by injection (yeah molbio!) to introduce this fusion protein into the worms. GFP Synaptobrevin

2008 Nobel Prize- GFP GFP mice

The pGLO plasmid ori- origin of replication GFP- green fluorescent protein bla- Beta-lactamase araC- Arabinose What are all the other marks? Why are they there?

Beta- lactam antibiotics have a similar structure Includes penicillin, ampicillin, and others The beta-lactam ring is a square structure common to all

Beta-lactamase can destroy a beta-lactam ring Breaking the ring destroys the antibiotic’s effectiveness

What about araC? Arabinose is a 5-carbon sugar, different from ribose