Biol215 Mid Session Quiz Study Questions

Week 1 – DNA as the Genetic Material + DNA Analysis

1. Describe the experiment that led to the notion of a ‘transforming principle’.

• Fred Griffith’s Experiment in 1928 provided the foundation of the discovery that DNA is the genetic material

• Griffith studied the infection patterns of the pathogen Streptococcus pneumoniae in mice, specially two major strains:

  • S-strain: colony on plate displayed a ‘smooth’ appearance due to a polysaccharide on its capsular surface

    • Provided protection from the host’s immune system

    • Virulent

  • R‐strain: ‘rough’ in appearance; lacking the polysaccharide

    • Easily destroyed by host’s immune system; avirulent  

  • Several variants of each major strain (IIS, IIIS, IIR, IIIR).

    • Occasionally IIS mutated to IIR, but never to IIIR. IIIS mutated to IIIR, but never IIR

1. Infection of Mice with Live Bacteria:

• Griffith injected mice with live Type IIR (alive, avirulent) bacteria. These mice remained healthy, no bacteria was recovered.

• He also injected mice with live Type IIIS (alive, virulent) bacteria. The mice developed pneumonia and died, IIS was recovered.

2. Heat-Killed Type S Bacteria:

• Griffith then injected mice with heat-killed (virulent) Type IIIS bacteria. The heat treatment killed the bacteria). These mice did not develop pneumonia and remained healthy. No bacteria recovered.

3. Combination of Heat-Killed Type S and Live Type R Bacteria:

• Then, Griffith injected mice with a mixture of heat-killed (virulent) Type IIIS bacteria and live (avirulent) Type IIR bacteria. The mice developed pneumonia and died, however IIIS was recovered.

2. What did Griffith’s experiments on the ‘transforming principle’ show?

• Griffith concluded that some heat-stable component present in IIIS transformed IIR → IIIS, and that it must have been inherited, as recovered cells remained IIIS when propagated

• Hence the term ‘transforming principle’

3. Describe the Avery, MacCleod and McCarty experiments.  What did these experiments establish?

• In 1944, Avery, MacCleod and McCarty repeated Griffith’s experiment but fractionated the ‘transforming principle’ in order to identify it.

• They heat-killed S cells, and then treated them chemically to purify the substance that transformed R cells → S cells

  • No treatment: R → S 

  • RNase treatment: RNA destroyed, R → S

  • Proteinase treatment: Protein destroyed, R → S

  • DNase treatment: DNA destroyed, R → R (no transformation)

    • Thus, DNA was likely responsible for the transformation, however they couldn’t rule out that low levels of contaminating protein were causing transformation.

4. Describe the differences between the assay used in the Griffith's experiment and the Avery et al. experiment? Why did Avery et al. change the assay that Griffith developed?

• Griffith used live R and heat-killed S bacteria, separately and in combination which he injected into mice

• Avery et al. used heat-killed S cells which they chemically treated and purified before adding to live R cells

• They changed the assay in order to identify the ‘transforming principle’ by fractionating it and chemically treating then purifying it.

5. How did Hershey and Chase differentially label protein and DNA, and how was this used to identify the genetic material?

• Hershey and Chase (1953) followed the passing of genetic material from parent to offspring T2 phages, by differentially labelling DNA and protein, by growing in media containing either 32P or 35S.

  • T2 phage produced in presence of 32P: 32P is primarily in the DNA, small amounts in protein components

  • T2 phage produced in presence of 35S: 35s is primarily in the protein elements, small amounts in DNA components

• Experiment 1: the phage were grown in the presence of 32P/35S, but bacteria that were not, thus the only source of the radiolabel was the phage. Phage infected cells, then blended and centrifuged out.

  • 32P grown T2: 32P was mostly found in the pellet with cells, thus it transferred with the DNA

  • 35S grown T2: 35S was mostly found in the supernatant with bacteriophage protein coat, thus it was not transferred and protein was not the genetic material 

  • This confirmed that DNA was the only material to enter cell → genetic material

• Experiment 2: similar, except the phage were given time to reproduce in bacteria, offspring were then harvested and examined for radioactivity.

  • When examined, small amounts of 32P (DNA) were found in the new phage, but no 35S (protein). DNA was passed on, not protein, thus identifying DNA as genetic material

6. Describe using diagrams the experiments with TMV that led to the discovery of RNA as genetic material for some viruses.

• 1956 Gierer and Schramm: RNA was identified as the genetic material in TMV virus.

• 1957 Fraenkel-Conrat and Singer: confirmed G+S findings

7. How does viral genetic material differ from prokaryotes and eukaryotes?

8. Describe the packaging of DNA in Eukaryotes.

9. Outline the mechanisms prokaryotes use to package DNA.

10. Describe the steps you would need to take to extract genomic DNA from a bacterial culture.

• Centrifugation is performed between each step to pellet various components 

1. Lyse cells with physical/ chemical force to disrupt cell membranes (detergent)

2. Remove cell contents by treating with protease to destroy protein and RNase to destroy RNA and precipitate for removal by centrifugation

3. Precipitate DNA in the supernatant with isopropanol and wash in ethanol 

4. Resuspend in water or buffer to concentrate

11. Describe how DNA migrates in agarose when subject to electrophoresis.

• DNA (negatively- charged due to phosphate backbone) travels towards the positive anode based on size. The smaller the DNA fragments, the faster it travels.

12. Outline two methods of analysing extracted genomic DNA – what information about the sample does each provide?

• Spectrophotometry: 

  • Check absorbance at 260/280 nm for approximate concentration and indication of purity 

• Agarose gel electrophoresis:

  • Visualisation of extracted DNA to distinguish DNA fragments of different lengths

13. Answer the following in relation to restriction enzymes.

    1. What do they do in nature?

• Recognise and cut the sugar phosphate backbone of DNA at specific sequences to create smaller fragments

• Catalyse double stranded DNA breaks 

• Can produce ‘sticky ends’ or ‘blunt ends’

2. How are they named?

• 1st letter of capital genus name + 2 letters of species name (italics) + 1st letter of strain name + roman number (order of RE identified from same strain)

• Eg. EcoRI → Escherichia coli Ry13

14. Discuss the components of a Sanger sequencing reaction.

• Template DNA

• Sequencing primer

• DNA polymerase

• Nucleotides (dNTPs: dATP, dCTP, dGTP, dTTP)

• Dideoxynucleotides (ddNTPs)

15. Describe the Sanger sequencing method. Sanger sequencing

• The Sanger sequencing reaction needs to occur for all four bases and results in fragments of varying lengths

1. The template DNA is first denatured into single strands using HEAT

2. An oligonucleotide (short DNA strand) called a “primer” is annealed to one of the two DNA strands.

• The primer is usually 10‐20 nucleotides long. It is designed by the investigator so that it’s 3’ end is next to the DNA sequence of interest

3. The primer “primes” DNA synthesis which is catalysed by a DNA polymerase enzyme

•  The 5’ to 3’ orientation ensures that the DNA made is complementary to the original sequence of interest

4. DNA polymerase + the four regular deoxynucleotide precursors are added: dNTPs: dATP, dTTP, dCTP, dGTP

5. PLUS small amount of dideoxynucleotides (modified nucleotide precursors) ddNTPs: ddATP, ddTTP, ddCTP, ddGTP 

• has a 3'H rather than a 3'‐OH on the deoxyribose sugar

• also have fluorescent dyes that allow detection (modern)

6. DNA polymerase adds a nucleotide to the 3’OH at the end of the primer 

7. Two possibilities:

   1. Since most of the DNA precursors in the reaction are dNTPs, the probability is high that a dNTP will be used for the next extension step.

   2. BUT there’s a small chance that a ddNTP is used... and then the extended DNA chain has a 3’‐H at the end, and DNA polymerase can’t add any more nucleotides. SO ddNTP “terminates” the DNA synthesis reaction

8. Fragments are then separated by gel electrophoresis

• Laser eye at end of capillary detects coloured fragments, 

• Computer then converts minor colour differences into more obvious differences that allows output of coloured peaks corresponding to each nucleotide position.

16. Compare and contrast the traditional Sanger sequencing method with the newer high throughput technology.

• Traditional methods used only 1 ddNTP in 4 different reaction tubes, and analysed using autoradiograph

• Modern method uses 4 fluorescently labelled ddNTPs in only one reaction, PCR is used for making sequencing templates and uses automated machines attached to computers for data analysis

  • Sequences millions of DNA fragments simultaneously → megabases and gigabases

  • Cost per megabase of DNA reduced significantly 

17. What is a BLAST search and when would you use it?

• A BLAST search is an online database tool used to find regions of similarity between biological sequences ie. for gene identification following sequencing

• It is best used for protein sequences

18. What is ORF finder and when would you use it?

• ORF finder is an open reading frame finder

• You would use it to search for open reading frames and translate amino acid sequences prior to database similarity searching and when cloning for protein expression

19. Outline the components required for amplification of DNA.

• Template DNA (free from nucleases)

• Primers (gene specific): forwards and backwards

• DNA Polymerase (heat stable)

• dNTPs (incorporated into new chains)

• Polymerase Buffer (enzyme specific, contains salts, cofactors (Mg2+) etc.)

20. Describe the thermocycling stages of a PCR reaction.

1. Denaturation (95º): heat sample to separate dsDNA to ssDNA

2. Annealing (45-55º): cool the sample to allow single stranded DNA primers to bind to separated ssDNA strands

3. Extension (72º): heat sample to allow Taq polymerase to extend primers (5’-3’) to synthesis 2 new DNA strands

21. Outline the steps performed in a Southern blot experiment.

• DNA fragments from an agarose gel are transferred to a filter by “blotting” and then detected using a homologous DNA probe which has been labelled

1. Regular agarose gel electrophoresis of DNA fragments

2. ‘Blot’ DNA fragments to transfer from agarose gel onto membrane 

3. Hybridise membrane (in buffer solution) with labelled probe

4. Wash away excess probe and detect target DNA 

22. How does a Northern blot differ from a Southern blot?

• Northern Blotting detects gene expression

• RNA is transferred to a membrane by ‘blotting’ and then detected using a homologous DNA probe which has been radioactively/ fluorescently labelled

23. Describe DNA microarray technology and provide an example of its use.

• Microarrays allow the analysis of thousands of genes in a single experiment and is based on hybridisation of a known probe with sample DNA

• On the surface of the chip, there are thousands of spots which contain a gene probe for a known sequence or gene

• Allows detection of mutations

• Eg. detect biomarkers for cancer and point mutations

Quiz

1. Traditional Sanger sequencing produced the following gel results

What would the 5' to 3' sequence be?

1. CCTAGCCGA

2. GGCCCCAAT

3. GGATCGGCT

4. AGCCGATCC

5. None of the above sequences are correct

2. Automated DNA sequencing is an improvement of traditional Sanger methods where

   1. ddNTP’s are used for chain termination

   2. Fluorescently labelled dNTP’s are used for chain termination

   3. Fluorescently labelled ddNTP’s are used for chain termination

   4. PCR is used for making sequencing templates

   5. Both c) and d) are correct

3. The most likely source of Taq polymerase used in PCR is a bacterium that lives in

   1. soil

   2. hot vents (heat stability)

   3. arctic ice

   4. humans 

   5. plants

4. Which of the following is not true about a linear molecule of double-stranded DNA?

   1. It is a double helix composed of antiparallel strands.

   2. Bases are paired via hydrogen bonds.

   3. At one end, two 5′ phosphate groups can be found.

   4. At one end, a 3’ hydroxyl group can be found.

   5. Pentose sugars are linked via covalent phosphodiester bonds.

5. Chromatin is best described as

   1. nucleosomes supercoiled around each other

      1. Nucleosomes: DNA wrapped around histones

   2. a DNA strand wrapped around histone proteins

   3. one strand of supercoiled DNA double-helix

   4. one fully packaged DNA molecule

   5. supercoiled and looped DNA compaction

6. In Hershey & Chase’s second experiment, where T2 phage were radioactively labeled with either 35S or 32P, and allowed to infect and reproduce in E. coli, they were able to demonstrate

   1. both 32P and 35S were found in progeny phage.

   2. only 32P was found in progeny phage.

   3. only 35S was found in progeny phage.

   4. no 32P was found in progeny phage.

   5. Both c) & d).

Week 2 – Genetics of Bacteria and Viruses + Introduction to Plasmids

1. Outline the similarities and differences between chromosomal and plasmid DNA

2. Explain the differences in procedure to extract chromosomal DNA from a bacterial cell as opposed to plasmid DNA

3. Describe three virulence determinants that promote colonisation in pathogenic bacteria

1. Ability to use motility and other means to; 

   1. contact host cells and disseminate within a host 

   2. combat ciliary action of host epithelial surfaces

2. Ability to adhere to host cells and resist physical removal

   1. Pili

   2. Afimbrial adhesins 

3. Ability to invade host cells

   1. Production of enzymes for digestion of host membranes 

4. Ability to evade host defences

   1. Capsule and slime production

4. Define the microbiome and outline 4 benefits it has to human health

• Microbiome: the microorganisms that live in or on the human biome which contribute to health

1. Help digest food

2. Regulate immune system

3. Protect against other bacteria that cause disease

4. Produce vitamins eg. B12, thiamine, riboflavin, vitamin K

5. What is horizontal gene transfer? Describe the different methods.

• Horizontal Gene Transfer: the transfer of genetic material between organisms of different species.

1. Conjugation: unidirectional transfer of genetic information via cell-to-cell contact between donor and recipient

   • Requires physical contact, mediated by conjugation pili, 1) F+ donor pilus draws cells together and forms cytoplasmic bridge.

2) One strand of F plasmid DNA transfers to F- recipient

3) Recipient synthesises complementary strand to become an F+ cell with a pilus (transconjugant), and donor cell synthesises complementary strand to restore F plasmid

• F+ cells have F factor: plasmid = donor cells

• F- cells lack plasmid = recipient

• Recipients that have received DNA = transconjugants

  1. Davis U-Tube experiment concluded contact requirement

2. Transformation: unidirectional transfer of extracellular DNA into recipient cells

   • Cells may have natural or induced competence (ability to uptake DNA)

   • DNA may be integrated into transformants’s genome or exist as a plasmid 

   • Recipients that have received DNA =  transformants

3. Transduction: the process in which a bacteriophage transfers genetic material from one bacteria to another 

6. Discuss natural and induced competence in bacteria

• Competence: physiological state permitting efficient uptake of macromolecule DNA

• Majority of bacteria are not naturally competent, however ability to uptake DNA varies between species

• Competence can be induced with chemicals (heat shock) or electric shock → temporary pores in membrane for uptake of DNA

7. Describe the experiment that determined conjugation required physical contact between cells

• Conjugation was discovered in 1946 by Lederberg + Tatum

• They studied two E. coli strains that differed in their nutritional requirements

  • Strain A: met, bio, thr+, leu+, thi+

  • Strain B: met+, bio+, thr, leu, thi

• Auxotrophic – cells cannot make certain nutrient required for growth

• Prototrophic – cells are able to synthesise all nutrients

• Minimal medium – contain the minimum nutrients possible for colony growth, generally without the presence of amino acids

1. Plated strain A on minimal medium: no colonies

2. Plated strain B on minimal medium: no colonies

3. Plated mixture of strain A and B on minimal medium: prototrophic colony growth = DNA combination and transfer = conjugation

• Conclusion: bacterial cells could transfer genetic material directly from one cell to another through a process now known as conjugation.

8. What is transduction and how does it contribute to genetic recombination and evolution in bacteria?

• Transduction: the process in which a bacteriophage transfers genetic material from one bacteria to another 

• Genes from a host cell are incorporated into a bacteriophage and then carried to another host cells when the bacteriophage initiates another cycle of infection

1. Phage infects the donor bacterial cells

2. Phage DNA and proteins are made, bacterial chromosome is broken down into pieces

3. Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then donor cell lyses and releases phage particles containing bacterial DNA

4. Phage carrying bacterial DNA infects a new host cell, the recipient cell

5. Recombination can occur, producing recombinant cells with a genotype different from both the donor and recipient cells.

9. How do Gene Transfer Agents differ from bacteriophage?

• Gene Transfer Agents: DNA containing virus-like particles that are produced by some bacteria

• Encoded by a cluster of head, tail and DNA packaging genes strongly resembling those of bacteriophage

• DNA fragments are packaged and injected into cells and therefore mediating HGT

• GTA’s have an increased capacity for genotype change in recipient cells compared to bacteriophages

  • GTA particles carry random DNA fragments from host cell → change genotype

  • Phages carry phage OR host DNA → recipient may become infected OR rarely change genotype

10. What is a High Frequency Recombination strain of bacteria, how is it produced and how does it differ in the transfer of genetic material to an F+ strain?

• High Frequency Recombination Strain: strains of F plasmids (episomes) that can transfer chromosomal genes, not just plasmid genes, into the chromosome

• Episomes: any plasmid that can integrate into the chromosome 

1. F+ cell undergoes integration of F factor into chromosome via crossing-over → Hfr cell

2. Conjugation of Hfr with F- cell

3. Integration of F factor is nicked, and nicked strand transfers to the recipient cells, bringin bacterial genes with it

4. Transferred strand is replicated, and donor bacterial genes appear in the recipient → recombination between transferred donor chromosome and recipient chromosome

11. Describe the general features of a plasmid and two additional features that are specific to recombinant DNA technology for the purpose of expression.

• Plasmid: small, circular, double-stranded DNA molecule that replicates independently 

  • Multiple cloning site: unique restriction sites for insertion of foreign DNA (cloning)

  • Origin of replication: sequence needed for plasmid to replicate in host

  • Selectable marker: cells with plasmid are easily distinguishable, from cells that don’t, usually antibiotic resistance genes

  • Infectious: self-transmissible via conjugation

  • Can integrate into the main chromosome: episome

12. What is the link between resistance plasmids, horizontal gene transfer and multi-drug resistant strains of bacteria

• Resistant plasmids can carry multiple antibiotic resistant genes, which are typically transferred by conjugation (HGT), and responsible for multi-drug resistant pathogenic strains of bacteria.

13. Using a specific virulence plasmid as an example outline two features/genes on the plasmid and how they contribute to virulence

• Virulence plasmids: encode genes that help bacteria infect organisms

• Virulence factors:

  • Toxins that damage or kill cells

  • Help bacteria attach to and invade cells

  • Protect bacteria against retaliation by immune system 

14. What is a bacteriocin and what characteristic gives it the potential to act as a food preservative?

• Bacteriocin: proteinaceous toxin, carried by bacteriocin plasmids

  • Can kill bacterial cells of the same or similar species that lack the plasmid → can kill unwanted bacteria to preserve foods

15. Describe the various forms plasmid DNA can assume and how this affects their migration when subject to agarose gel electrophoresis

• Open circle - slowest

• Supercoiled - fastest

• Linear - slightly faster than open circle

• Dimer - slower than all single plasmids

16. Describe viral genomes

• Viral genomes: the total genetic content of a virus (very small)

  • contain a single type of nucleic acid (DNA OR RNA)

  • single stranded OR double stranded

  • linear OR circular

17. Outline how genetic material is replicated in retroviruses

1.  Reverse transcriptase makes a DNA copy of the RNA degrading the RNA at the same time

2. Then uses this DNA strand as a template to complete a DNA double helix

3. The DNA then enters the nucleus and integrates into the chromosomal DNA of the host – becoming a PROVIRUS

4. The proviral DNA is transcribed into viral RNA fragments and translated into viral proteins

5. New capsids are assembled around viral RNA fragments and reverse transcriptase

6. The nucleocapsid “bud” from the plasma membrane as complete virus

18. Outline the reasons behind the high mutation rate observed for the HIV virus

• HIV has a high mutation rate due to the very high replication rate, and the high error rate of the virion reverse transcriptase 

19. How do viruses contribute to cancer? Provide a specific example in your answer

• Some viruses carry oncogenes as part of their genomes, and some influence the expression of proto-oncogenes or suppressor genes already present in the host

• Eg. Cervical cancer can be acquired through the human papillomavirus (HPV).

Quiz

1. With respect to gene transfer, what is transduction in bacterial genetics?

   1. The transfer of genetic material from a bacterium to a virus.

   2. The process of bacteria producing their own viruses.

   3. The exchange of genetic material between bacteria through direct cell-to-cell contact.

   4. The transfer of bacterial DNA from one bacterium to another via a bacteriophage 

   5. The mechanism by which bacteria acquire energy from their environment.

2. What are the plasmid status of bacterial cells resulting from conjugation between a F+ and a F- bacterium?

   1. Two F+ bacteria

   2. Two F- bacteria

   3. The F+ bacterium becomes F- , and the F- bacterium becomes F+

   4. The F+ bacterium remain as F+ , and the F- bacterium remain as F-

   5. The F+ bacterium becomes Hfr, and the F- bacterium becomes F+

3. The first demonstration of recombination of DNA between bacteria was achieved by:

   1. Lederberg and Tatum

   2. Luria and Delbruck.

   3. Griffith

   4. Davis

   5. Watson and Crick

4. Which of the following components is brought into a cell by HIV?

   1. DNA-dependent DNA polymerase.

   2. RNA polymerase

   3. Ribosome

   4. Reverse transcriptase

   5. Both b and d are brought into the cell by HIV

Week 3 – Recombinant DNA Technology and Recombinant Proteins

1. Answer the following in relation to restriction enzymes (REs).

   1. For what purpose are they used during cloning?

• Recognising and cutting the sugar phosphate backbone of DNA at specific sequences to create smaller fragments

• Allow target gene to be cut and then ligated into vector for cloning

2. What do they recognise?

• Specific short palindromic sequences in DNA. 

3. What kinds of products do they generate?

• Can produce 5’ ‘sticky ends’ (P overhang), 3’ ‘sticky ends’ (OH overhang) or ‘blunt ends’

2. What is colony PCR, and how do you use it in recombinant DNA technology?

• Screening method to check for successful cloning

1. Design primers to detect your insert

   1. Insert-specific primer: complementary to insert

   2. Backbone-specific primer: complementary to vector

   3. Orientation-specific primer: complementary to insert and vector- must face each other

2. Run a PCR reaction using supernatant of lysed bacteria as template

3. Run in AGE to analyse product size

3. What are antibiotics used for in recombinant DNA technology?

• Selection/ screening to identify clones carrying gene of interest

• Positive selection: antibiotic resistance

4. What are the mechanisms and advantages of each one of the cloning techniques discussed during the lectures?

5. What are the various directional cloning methodologies?

Week 4 – Genetic Regulation in Bacteria

1. What are the general/common features of operons?

• Operon: group of genes transcribed from the same promoter

  • 1 Promoter

  • Group of genes

  • Transcription terminator

• Some operons also contain:

  • Operator + regulatory genes

  • Constitutive Genes: genes expressed at similar levels at all time under all conditions – not regulated

  • Regulated Operons: contain genes that are switched on/off only after induction

2. What are the protein products of the lac operon, and their functions?

• B-galactosidase → lactose hydrolysis (lacZ)

• Lactose permease → lactose transport (lacY)

• Transacetylase → unknown, cellular detoxification?? (lacA)

3. What is catabolite repression and its genetic regulation mechanism on the lac operon?

• cAMP binds to catabolite activator protein (CAP) → forms cAMP-CAP complex → binds to CAP site in lac operon → recruits RNA polymerase to promoter → increases rate of transcription – positive regulation of lac operon

  • Glucose inversely proportional to cAMP = lac

  • Glucose reduces cAMP → reduces lac

4. How does the attenuator/leader peptide work in regulating the trp operon?

• Low trp → trp genes expressed BUT below maximal levels by controlling ratio between long mRNA and short mRNA

• Attenuation: the process to terminate transcription early to form short mRNA

• Attenuator: part of RNA sequence that forms 2ndary structures and governs level of transcription of attenuated operons

• trpL gene is transcribed into leader mRNA, which contains a short open reading frame for leader peptide and attenuator – consisting of 4 regions with complementary sequences

• Pairing of:

  • Regions 1 and 2 – Transcription pause signal (ribosome loaded and translation follows)

  • Regions 2 and 3 – Anti-termination signal (allows transcription to continue)

  • Regions 3 and 4 – Termination signal (stops further transcription)

• High trp → ribosome runs through region 1 quickly and occupies 2 → prevents 2 from pairing with 3 → region 3 pairs with 4 → transcription termination signal → produces short mRNA → no trp genes transcribed

5. What kinds of genetic regulation(s) operate on lac and trp operons?

6. Compare and contrast the genetic regulation mechanism(s) of the lac and trp operons

Promoter: transcription initiation sequence (DNA) in front of RNA start site

Operon: group of genes transcribed from the same promoter

Repressor: molecule that blocks transcription from the promoter – binds to operator → negative regulation

Activator: molecule that increases gene transcription of a gene or set of genes – binds to activator-binding site → positive regulation

Operator: the site (DNA) at which the repressor binds to block transcription

Inducer: molecule that induces transcription from the promoter

Induction: synthesis of gene product(s) in response to an inducer

Attenuation: the process to terminate transcription early to form short mRNA

• LacI gene (outside of lac operon) → produces lac repressor proteins → tetramer → attaches to operator and prevents transcription → genes not expressed

• Allolactose: lac operon inducer

  • Negative control of lac operon

  • Active repressor → constitutive lacI repressor

  • Inactive repressor + Allolactose inducer → allosteric shift → inactive repressor → cannot bind to operator → activation of lac operon

• High trp → trp binds to trp repressor → activated repressor binds to operator → trp genes regulated

• No trp → repressor inactive → trp operon expressed

Quiz

1. Genes required for metabolising the sugar lactose are constitutively expressed in E. coli.

   1. True

   2. False

2. What consists of a group of genes but only one promoter and usually only one transcription terminator at the end, and usually found in prokaryotes?

   1. Operon

3. LacI gene encodes for the repressor protein that ...

   1. binds to lactose

   2. regulates the genes found in the lac operon

   3. forms a tetramer

   4. binds to glucose

   5. binds to allolactose

4. The genes in the lactose operon are turned on as soon as lactose becomes available, even if there is also glucose present.

   1. True

   2. False

5. Tryptophan operon is directly regulated at the genetic level by ...

   1. two mechanisms

   2. attenuation

   3. repression

   4. feedback inhibition of anthranilate synthetase by tryptophan

   5. the presence or absence of tryptophan