Microbial genetics
What are the types of RNA-based regulation?
(i) ANTISENSE RNA - Small RNAs with nucleotide sequence complementary to a specific mRNA sequence (used to tun off expression of genes) - Base pairing (~30 nucleotides) between these RNAs prevents translation - Transcribed by a separate gene - Recognise multiple mRNAs that have the same target sequence - PREVENTS TRANSLATION (ii) RIBOSWITCHES - When RNA acts as repressors or activators RNA can act in a similar fashion to protein: can interact with effector molecules - Binding of the metabolite/effector molecule to RNA determines its structure, which in turn influences gene expression - Result in change in production of proteins encoded by the mRNA
How does AraC compare to lacI?
(i) AraC ALWAYS BOUND NEAR THE ara DNA REGION - Allows for rapid changes in ara gene expression (on or off) in response to environmental signals (presence/absence of arabinose) (ii) LacI not always associated with DNA - Re-establishing repression of the lac operon takes more time - Less efficient form of regulating gene expression
Explain how the mal regulon is regulated depending on whether arabinose, glucose, both or neither is present.
(i) Arabinose PRESENT, AraC acts as ACTIVATOR - Compact AraC dimer forms which recognise araI1 and araI2 - DNA loop is broken - Promotes CRP and RNAP binding - Expression of ara genes (ii) Arabinose ABSENT, AraC acts as a REPRESSOR - Elongated AraC dimer forms which recognises araI1 and araO2 - DNA loop formation - In this form, AraC cannot recruit the RNAP and the CRP binding site is blocked - No expression of ara genes
Describe the types of bacterial gene expression.
(i) CONSTITUTIVE GENE EXPRESSION - Genes are always 'ON' i.e. always expressed/transcribed - Normally genes whose products are always required under all conditions E.G. polymerase genes, ribosomal proteins, housekeeping genes (cells that mediate essential functions of the cell) (ii) INDUCIBLE GENE EXPRESSION - Genes are TURNED ON (transcription occurs) in response to an environmental signal - Induced by the presence of an effector molecule/signal (inducer, process occurs through induction) - Inducible genes are normally those which encode enzymes involved in catabolic (degradative) processes E.G. Lac operon = genes involved in breakdown of lactose are turned on in the presence of lactose (iii) REPRESSIBLE GENE EXPRESSION - Genes are TURNED OFF (transcription is blocked) in response to an environmental signal - Repressed by the presence of an effector molecule/signal (co-repressor/inhibitor, process occurs through repression) - Repressible genes are normally those which encode enzymes involved in anabolic (biosynthetic) processes E.G. amino acid biosynthesis operons are not expressed if there are already high levels of that amino acid present
What are two kinds of bacterial genomes?
(i) CORE GENOME - Shared by all strains of the species - Essential genes for cell function (ii) PAN GENOME - Not shared by all strains of the species - Additional genes not essential for the species (e.g. plasmids and genomic islands)
Identify the features regulatory proteins require.
(i) EFFECTOR MOLECULE BINDING SITE (ALLOSTERIC SITE) - Site where the effector molecule interacts with the protein to change its structure (and thus function) - If a gene encoding a regulatory protein is mutated such that the amino acids at the effector binding site change, the AFFINITY for the effector molecule is affected - Stronger affinity -> stronger expression (ii) PROTEIN-PROTEIN INTERACTION DOMAIN - For homo-dimerisation of the protein so it can bind to DNA - Can also be required for interacting with other proteins (e.g. interaction between activator protein and the RNAP) (iii) DNA BINDING DOMAIN - Often sequence specific - specific interactions between amino acid side chains and chemical groups on nucleotides - Mutations that change the amino acids at the DNA binding domain can affect the ability of the protein to bind to DNA
How do you identify a PI?
(i) G+C content differs from the rest of the genome (ii) Insertion-type sequences at the ends of the island which were most likely used for integration into the recipient chromosome (iii) Flanking genes are often tRNAs (iv) Absent from non-pathogenic strains of the same species
Explain how the mal regulon is regulated depending on whether maltose, glucose, both or neither is present.
(i) Glucose ABSENT, maltose PRESENT - cAMP production, formation of CRP-cAMP complex to activate expression of malT - MalT + CRP-cAMP activate expression of mal genes (ii)Glucose ABSENT, maltose ABSENT - cAMP production, formation of CRP-cAMP complex to activate expression of malT - MalT cannot bind to malT box due to absence of maltose -> no expression of mal genes (iii) Glucose PRESENT, maltose PRESENT - No production of cAMP or malT - No expression of mal genes (iv) Glucose PRESENT, maltose ABSENT - No production of cAMP or malT - No expression of mal genes
Identify the types of DNA binding motifs.
(i) HELIX-TURN-HELIX MOTIF - Two alpha-helices in the protein which interact and promote DNA binding - The recognition helix recognises the sequence via hydrogen bonds - Very common in bacteria DNA binding proteins (ii) ZINC FINGER MOTIF - Most common on eukaryotic regulatory proteins - Specific amino acids bind zinc which promotes correct folding of the DNA binding structure (iii) LEUCINE ZIPPER MOTIF - Does not interact with the DNA itself - Series of leucine residues spaced at regular intervals - Interaction between the leucine side chains hold the recognition helices in place for DNA binding - Most common in eukaryotic proteins
Describe the steps involved in the expression of bacterial gene(s). Transcrition.
(i) INITITIATION = The DNA molecule unwinds and separates to form a small open complex. RNA polymerase binds to the promoter of the template strand. (ii) ELONGATION = Sigma factor recognises promoter. RNAP (holoenzyme) synthesises an mRNA molecule. (iii) TERMINATION = Rho-dependent termination (protein "Rho" is responsible for disrupting the complex of template strand, RNAP and RNA molecule); OR Rho-independent termination (loop forms at the end of RNA molecule, causing it to detach itself)
What are the types of RNA molecules involved in transcription (gene expression)? Describe briefly.
(i) Messenger RNA (mRNA) - Encodes information for synthesis of protein - Translated (ii) Ribosomal RNA (rRNA) - Structural component of ribosomes - Not translated (iii) Transfer RNA (tRNA) - Adapter molecules (between mRNA and ribosome) - Carry amino acids to the ribosome for the translation of mRNA - Not translated
How does the translation of trpL (leader peptide) occur at no/low levels or high levels of tryptophan?
(i) NO/LOW LEVELS - Causes ribosomes to stall at trp codons (waiting for trp amino acids); ribosome is covering region 1 RNAP continues transcription: - Once transcribed, region 2 cannot base pair with region 1 but is free to base pair with region 3 - Termination hairpin (region 3:4) cannot form (ii) HIGH LEVELS - Transcription has been initiated - Ribosome translated leader and pauses at trpL stop codon (regions 1 and 2 are covered) RNAP continues transcription: - Region 2 is no longer free to base pair with 3 - Once again 4 is transcribed, region 2 is free to base pair with it - RNAP fall off and transcription is terminated
Explain how the trp operon is regulated depending on whether tryptophan is present or absent.
(i) PRESENT - Once tryptophan accumulates, it binds to the aporerepressor - Holorepressor binds to operate preventing RNAP binding - Transcription doesn't occur (or low level expression) (ii) ABSENT - As a aporerepressor, trpR can't bind to operator - Promoter is free for RNAP to bind and initiate transcription of trp genes
Describe the mechanisms by which bacteria can terminate transcription.
(i) RHO-DEPENDENT TERMINATION - mediated by Rho protein - Attaches to GC rich regions and moves along the RNA, following the RNAP - RNAP encounters a terminator sequence causing it to pause - Allows Rho to catch up to the RNAP and unwind the DNA-RNA (ii) RHO-INDEPENDENT TERMINATION - does not involve the Rho protein - RNA moves past inverted repeats (IR) and transcribes termination sequence - IR arrangement creates a hairpin loop structure - Loops makes the RNAP slow down and eventually stop
What are the two proteins of the two-component regulatory system?
(i) SENSOR KINASE (SK) - Kinase = enzyme that can phosphorylate (transfer phosphate group) itself or other proteins - Membrane-bound - Senses external signal - Presence/absence of external signal causes SK to phosphorylate itself at a histidine residue (autophosphorylation) - Phosphate then transferred to RR (ii) RESPONSE REGULATOR (RR) - Phosphorylation will either activate or inactivate RR STEPS 1- SK detects condition outside of the cell 2- Signal triggers (or prevents) phosphorylation 3- Phosphate is transferred to a cognate RR in the cytoplasm. Regulator binds DN and either stimulates/represses the target genes. 4- A phosphatase removes the phosphate and down-regulates the system.
What is trpL? How is it determined which structure is formed?
- Leader peptide (14 amino acids) is tryptophan rich - Contains 4 sequences (region 1-4) capable of forming secondary structures REFER TO NOTES FOR STRUCTURES (i) BASE PAIRING REGIONS OF 3 + 4 - Forms a termination hairpin (like rho-independent termination) - Transcription is terminated - Regions 1 + 2 can also base pair (ii) BASE PAIRING REGIONS OF 2 + 3 - Termination hairpin cannot form - Transcription into trp genes proceeds trpL FORMS IS DETERMINED BY: - Level of tryptophan in the cell - Behaviour of the ribosome in translating the leader peptide in response to tryptophan levels ------ (i) LOW trp levels - Ribosome translates slowly, will pause and cause formation of anti-terminator (non-terminating hairpin) - Prevents formation of terminator and allows transcription to continue (ii) HIGH trp levels - Ribosome translates quickly, will fall off the mRNA after translating leader peptide - Allows terminator hairpin to form, RNAP detaches and transcription ends
Describe the process of transformation.
1- DNA binds to recipient cell 2- Membrane bound proteins bind to DNA 3- Import of one strand of DNA into the cell 4- Competence proteins bind to DNA (protect DNA from nuclease) 5- Homologous recombination and RecA mediated
Describe how two-component regulatory systems work.
1- SK detects condition outside the cell. 2- Signal triggers (or prevents) autophosphorylation. 3- Phosphate is transferred to a cognate response regulator in the cytoplasm. Regulator binds DNA and either stimulates or represses the target genes.
Differentiate between riboswitches and attenuation.
A riboswitch is an RNA molecule that has folded up on itself to form a specific structure (sort of like a protein does but not nearly as complex). This RNA molecule can potentially bind to other RNA molecules in the presence of a cofactor (or without in some cases) and inhibit (or attenuate ) translation of the target RNA. Riboswitches can be attenuators, but sometimes they serve to relieve inhibitory mRNA secondary structures and activate translation.
How is the ara regulon regulated?
AraC forms a dimer: (i) When BOUND TO ARABINOSE, N-terminal binds to C terminus of the same molecule - Compact araC binds to araI1, and araI2 interacts with RNAP to permit araBAd transcription -> arabinose is catabolised (ii) WITHOUT ARABINOSE, N terminus binds to C terminus of other AraC subunit - Elongated araC bind at araI and araO2 and the DNA forms a loop
Explain the blue/white screen process used in molecular cloning.
BLUE colonies do not have vector with foreign DNA inserted WHITE colonies have foreign DNA inserted
Describe the process of plasmid mobilisation.
Contain: - Origin of transfer: oriT - Mob of genes and coupling protein: Dtr (donor transfer replication) Mobilisable plasmids are mobilised if Tra functions are provided in trans - pilus genes
What are regulatory proteins?
Control the expression of specific bacterial gene/operons Bind effector molecules or ligands which will result in their activation or inactivation Often expressed from a promoter that is separate from the gene that they target Function by binding to specific regulatory sequences adjacent to the promoter to modulate transcription
Explain the mechanisms of homologous recombination.
DEFINITION: the genetic exchange mediated by identical or almost identical DNA sequences ENDONUCLEASE cuts/nicks a single DNA strand in each molecule Displacement of nicked donor strand by DNA helicase & single stranded binding (SBB) protein binds RecA (recombinase) protein mediates base pairing between donor and recipient strands = CROSS STRAND BRIDGE Resolution of DNA molecules by RESOLVASE Mismatches are removed (EXONUCLEASE) and repaired (DNA POLYMERASE), followed by re-joining of DNA strands (DNA LIGASE)
What are transposable elements?
Definition: DNA elements that can move from one site in a DNA molecule to another; mobile DNA elements - "jumping genes" - Lack DNA replication site (must be part of a replicon i.e. csome or plasmid) - Recombinases (i.e. transposases, revolsases, integrases) - Random movement (mutagens) Types: (i) Insertion sequence (IS elements (ii) Transposons (more complex)
What is catabolite repression?
Definition: a global regulatory system used by bacteria to decided which carbon source to utilise first if more than one is present in a medium - Ensures glucose is exhausted first in a medium that contains more than one carbon source - Despite the presece of lactose, the lac operon is not expressed while glucose is still present
What are partial diploids?
Definition: bacterial strains that receive a second set of genes (e.g. lac operon) on a plasmid Said to be partial diploids as they have two copies of the gene in the lac operon, but only one copy of all other chromosomal genes Can be created using plasmids containing portions of chromosomal DNA - F' conjugation
Define and describe the basic characteristics of pathogenicity islands.
Definition: clusters (an 'island') of genes encoding virulence genes; part of the pan genome - Acquired by horizontal gene transfer - Sometimes genetically unstable (prone to rearrangments, deletions) PROPERTIES - Large (10-200kb) - Often have different G+C content (most likely reflects G+C content of the donor microbe) - Locations of PI include: csome, plasmids, phage genomes
Define and explain molecular/gene cloning.
Definition: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated MAIN STEPS OF GENE CLONING (i) Isolation and fragmentation of source DNA - Source DNA can be genomic DNA, RNA, or PCR-amplified fragments (ii) Insertion of DNA fragment into cloning vector - Most vectors derived from plasmids or viruses - DNA is generally inserted in vitro - DNA ligase: enzyme that joins two DNA molecules (iii) Introduction of cloned DNA into host organism - Transformation used to get recombinant DNA into host
What is attenuation?
Definition: mechanism of bacteria to prematurely terminate transcription Gene expression prevented AFTER TRANSCRIPTION is initiated, but BEFORE COMPLETE Number of completed mRNAs reduced, despite the fact the rate of initiation is not Sequence towards the 5' end of the mRNA can fold into ALTERNATIVE SECONDARY STRUCTURES - One secondary structure will allow transcription to proceed - Alternative secondary structure = attenuation - Form depends on behaviour of ribosomes ---- (Khan academy) This section lies between the operator and the first gene of the operon and is called the leader. - Encodes a short polypeptide and attenuator sequence - Attenuator does not encode a polypeptide, but when transcribed into mRNA, has self complementary sections and can form various hairpin structures
What are effector molecules? Give examples.
Definition: selectively binds to proteins; can increase or decrease activity, gene expression or cell signalling (i) INDUCERS - Presence activates transcription - Positively regulated - E.g. lactose as an inducer of the lac operon (ii) CO-REPRESSORS/INHIBITORS - Presence represses transcription - Negatively regulated - Tryptophan is a co-repressor for the trp operon
Explain how illumina sequencing (NGS) works and compares to sanger sequencing.
Definition: technique used to determine the series of base pairs in DNAT TARGETED NGS - High sequencing depth enables higher sensitivity - Higher discovery power - Higher mutation resolution - More data produced with same amount of input DNA - Higher sample throughput SANGER SEQUENCING - Fast, cost-effective for low numbers of targets - Familiar workflow
Define and describe conjugation and the steps involved.
Definition: transfer of genetic material between bacterial cells by direct cell-to-cell contact or through a sex pilus. PROPERTIES - F plasmid are large and cryptic - Narrow host range (i) Machinery (stages) in conjugation - Type IV secretion system (TRANSFEROSOME - build the sex pilus) - DNA processing complex (RELAXOSOME) - Coupling proteins (ii) Energetically expensive - Tightly regulated - F transfer at high efficiency because of a mutation in a repressor protein - Highly responsive to environmental stimuli
What is transformation? Name two types of artificial transformation.
Definition: uptake of naked DNA from the environment ARTIFICIAL INSEMINATION (i) Chemical transformations - Cells treated with calcium ion ad chilled - Exposed to elevated temperature (up to 30s) and DNA can be taken up - Not fully understood (ii) Electroportation - Cells exposed to high voltage electrical pulse which makes the cell permeable to DNA
Apply your knowledge of activator/repressor proteins and inducer/co-repressors to predict if an operon will be turned on or off.
Different bacterial regulatory systems System -> regulatory protein / effector molecule (i) +vely regulated, inducible -> activator / inducer (ii) +ve regulated, repressible -> activator / inhibitor (iii) -vely regulated, inducible -> repressor / inducer (iv) -vely regulated, repressible -> repressor / co-repressor REFER TO NOTES FOR EXAMPLES
Compare and contrast RNA polymerase in ekukaryotes, bacteria and archaea.
EUKARYOTIC Have three RNA polymerases responsible for transcribing different nuclear genes (i) RNA polymerase I = transcribes rRNA genes (in the nucleolus) (ii) RNA polymerase II = transcribes protein-encoding genes (mrNA) (iii) RNA polymerase III = transcribes tRNA genes BACTERIAL (As mentioned previously) ARCHAEA - One RNAP consisting of 11-12 subunits - Most similar to eukaryotic RNAP II - Recognises promoters that are similar to those recognised by eukaryotic RNAP II - Two transcription factors recognised conserved sequences in promoter and recruit RNAP
What makes glucose the most preferred carbon source?
Easy to degrade High energy yield Glucose catabolism genes are constitutively expressed (always on)
How does lactose get into the cell and turn on the lac operon?
Even when the lac operon is repressed, there is always basal level expression (i.e. there is always low level transcription) Low amounts of permease produced allow some lactose to enter the cell Low level of beta-galactosidase will convert lactose -> allolactose Allolactose can then bind to lacI and turn on the lac operon - lacZYA expressed at significantly higher levels
Name the basic subunits that make up the bacterial RNA polymerase.
Five subunits: α2ββ'ω (the core enzyme) (i) BETA (β) Ribonucleoside triphosphate binding site (ii) BETA PRIME (β') DNA binding abilities (iii) ALPHA (α) Required for assembly of the core enzyme (iv) OMEGA (ω) Function not well understood but involved in maintaining the core enzyme (v) SIGMA (σ) FACTOR Associated with the core enzyme to form holoenzyme (α2ββ'ωσ) and guides the complex to specific promoters to initiate transcription
Explain the ara operon. What is its regulatory protein?
Function: encodes catabolic enzymes required to break down arabinose - Arabinose -> xylulose 5-phosphate Regulatory protein = AraC - Acts as an ACTIVATOR in the PRESENCE of arabinose - Acts as a REPRESSOR in the ABSENCE of arabinose
What is the lac operon?
Function: encodes enzymes that are responsible for the catabolism (breaking down) of the sugar lactose so it can be used as a carbon source Not present in all bacteria - Common in non-pathogenic strains of enterics Not constitutively expressed Inducible system - presence of effector (inducer) stimulates expression Negatively regulated - lacI - Repressor protein - Binds to the lac operator sequence and blocks transcription of lacZYA
How is the expression of lac operon regulated?
IN THE ABSENCE OF LACTOSE - lacI repressor binds to the operator (regulatory sequence) and prevents the RNAP from binding - Transcription of the lac operon is turned off - Repressor lacI binds to lacO; the bound protein overlaps the lacZYA promoter (lacP) and engineers a DNA loop which prevents transcription IN THE PRESENCE OF LACTOSE - Lactose converted to allolactose -> binds to lacI (at the effector binding site) - Induces allosteric transition, changing the conformation of lacI so that it can no longer remain bound to the operator -> lacI falls off - Lac promoter is free for RNAP to initiate transcription
Define plasmid incompatibility and classification.
INCOMPATIBILITY When plasmids are unable to co-exist in a cell because of the same: - Replication mechanim - Copy number control - Partitioning mechanisms - IncP, IncN, IncF, etc. CELL CYCLE WITH 1 PLASMID 1- Cell grows and plasmid replicates 2- Cell division occurs 3- Each daughter cell has 1 copy of same plasmid CELL CYCLE WITH 2 INCOMPATIBLE PLASMIDS 1- Cell grows but plasmids do not replicate as 2 origins are already present 2- Cell division occurs 3- Incompatible plasmids have bee distributed to different daughter cells
Define integrons and explain how bacteria can use these elements to their advantage.
INTEGRONS: mobile genetic elements which that ability to capture genes (cassettes), notably those encoding antibiotic resistance, by site-specific recombination
Explain site-specific recombination.
In which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology
Identify and describe the three stages of bacterial transcription.
Initiation -> Elongation -> Termination INITIATION 1- RNA polymerase holoenzymes scans DNA for promoter sequence 2- Binding to the promoter sequence forms the closed complex 3- RNA polymerase unwinds DNA and begins transcribing RNA from ribonucleoside (rNTPs) 4- Sigma factor leaves the complex ELONGATION RNAP moves along the DNA template and extends the RNA strand (5'-3' direction) TERMINATION (i) Rho dependent termination - mediated by Rho protein - Attaches to GC rich regions and moves along the RNA, following the RNAP - RNAP encounters a terminator sequence causing it to pause - Allows Rho to catch up to the RNAP and unwind the DNA-RNA (ii) Rho independent termination - does not involve the Rho protein - GC rich sequence of inverted repeats which base pair when transcribed to form a stem loop - Stem loop formation causes RNAP to pause at a stretch of U:A base pairs (weak base pairing results in dissociation of the transcription complex)
How does catabolic repression work at the genetic level?
Involves +ve regulation Uses an activator protein = cycic AMP receptor protein (cRP Binds ad bends specific DNA sequences near specific promoters via a helix-turn-helix motif Cannot bind without its effector molecule cAMP
Apply your knowledge of the mal regulon to predict the consequences of different mutations.
MALTOSE CATABOLISM MUTANTS -> Resulting in ability to grow on media with maltose as a sole carbon source - malT^- = no functional malT protein produced - Mutation/deletion of malT box = can abolish or reduces expression of mal genes - Adenylate cyclase mutants
What are the basic rules of regulation of bacterial gene expression?
Mainly regulated at the transcriptional level Regulatory proteins can mediate this process by binding at the operator sequences - Repressor binding blocks transcription (-ve regulation) - Activator binding stimulates transcription (+ve regulation) Action of regulatory proteins are mediated by the presence/absence of effector molecules - Presence of an inducer promotes transcription - Presence of a co-repressor/inhibitor blocks transcription
What is the major regulatory protein of the mal regulon?
MalT: maltose activator protein (+ve regulation) - Regulates the expression of multiple mal genes - While the lac operon is -ve regulated by lacI, the mal regulon is +vely regulated by MalT - Cannot bind DNA without maltose (the inducer)
What is a two-component regulatory system?
Most common type of signal transduction system which uses two proteins to transmit the external signal - Sensor kinase (SK) - Response regulatory (RR) Effector remains outside of the cell (cannot get in)
Describe the basic features of bacterial genomes.
Most only have one chromosome and are haploid Covalently closed circular DNA molecules Genes are organised into clusters (operons) and lack introns Can also contain extra-chromosomal elements (plasmids) REFER TO NOTES FOR IMAGE OF E. COLI GENOME
What is a regulon?
Multiple operons involved in the same process (e.g. maltose fermentation) regulated by the same activator/repressor protein How does it differ from global regulation? Global regulators control the expression of unrelated genes involved in different processes (e.g. maltose, arabinose and lactose catabolism genes by CRP-cAMP)
Explain the TrpR repressor.
On its own = APOREPRESSOR - When TrpR is expressed and not bound to effector molecule - As a aporepressor, TrpR cannot bind to the trp operator sequence Requires a co-repressor = tryptophan (effector) Aporepressor + co-repressor = HOLOREPRESSOR - Binds to operator and inhibits transcription - Note: transcription is not completely repressed TrpR REPRESSOR FUNCTIONS AS A PART OF A REGULON - Can bind to >1 operator and regulate multiple sets of genes with related functions (i) TRP OPERON - Regulate the expression of enzymes needed for biosynthesis of tryptophan (ii) aroH GENE - Encodes an enzyme required in the early stages of aromatic amino acid (i.e. tryptophan) biosynthesis (iii) trpR - Binds to its own promoter to repress expression; autogenous regulation ------- (Khan academy) When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by physically blocking the RNAP. Trp repressor does not always bind to DNA, instead it binds and blocks transcription only when tryptophan is present -> trp repressor attaches to tryptophan and changes shape to become active. A small molecule like tryptophan, which switches a repressor into its activate state, is called a corepressor.
Define the basic features of a different cloning vector.
PLASMIDS AS CLONING VECTORS Natural vectors and have useful properties - Small size = easy to isolate - Independent origin of replication - Multiple copy number = many per cell - Presence of selectable markers Vector transfer carried out by chemical transformation and electroportation
(Regulatory proteins) Differentiate between positive and negative regulation.
POSITIVE - Binding of this protein to its regulatory sequence ACTIVATES transcription NEGATIVE - Binding of this protein to its regulatory sequence REPRESSES activation
Identify the major difference in the expression of prokaryotic and eukaryotic genes.
PROKARYOTES - Lack a nucleus therefore DNA floats freely - To synthesise a protein, transcription and translation occurs almost simultaneously - When resulting protein is no longer required, transcription stops; where more protein is required, more transcription occurs - Primary method to control expression is the regulation of DNA transcription; therefore, is mostly controlled at the transcriptional level EUKARYOTES - Have intracellular organelles that add to complexity - Processes of transcription and translation are physically separated by the nuclear membrane; DNA contained in nucleus where it is transcribed to RNA and transported out of nucleus -> cytoplasm where RNA is translated to protein - Transcription within nucleus; translation outside nucleus, in cytoplasm. - Regulation of gene expression occurs at all stages of the process
Describe the process of conductional transfer.
Plasmid A = non-mobilisable Plasmid B = conjugative Conjugation of cointegrate = conductional transfer Resolution can occur in recipient strain = non-mobilisable plasmid has tranferred
What is a plasmid? Describe the basic structural features.
Plasmids are DNA molecule that exist in an extra chromosomal state - contain their own oriV (origin of vegetative replication) UBIQUITOS - widely dispersed throughout bacteria; not all have plasmids and some have multiple SIGNIFICANT PHENOTYPES - pathogenicity determinants/antibiotic resistance/degradative ability - e.g. Bacillus anthracis (causes anthrax) - contains 3 plasmids that encode pathogenicity determinants; when lost, no longer pathogenic. EVOLUTIONARY SIGNIFICANCE - gene dissemination (transformation, transduction, conjugation and recombination) DISTRIBUTION - gram +ve and gram -ve bacteria SIZE AND COPY NUMBER VARIATION - Small plasmids (cryptic): no phenotype - ~20kb (non-conjugative): unable to transfer themselves - >30kb (conjugative): large number of conjugative genes - >100kb (megaplasmids) - occur in Agrobacterium - Vary in host range (broad vs narrow)
What are the genetic events that contribute to plasmid recombination events?
RECOMBINATION: rearrangement and exchange of genetic material between two sources RECOMBINATION MECHANISMS (i) Non-homologous recombination (ii) Homologous recombination (iii) Site-specific recombination
Explain how the lac operon is regulated depending on whether lactose, glucose, both or neither is present.
REFER TO DIAGRAM IN NOTES.
Apply understanding of partial diploid strains and the lac operon to predict the consequences for lac gene expression.
REFER TO EXAMPLES IN NOTES (IMPORTANT!)
Describe how RNA can be used to regulate gene expression.
REGULATION OF GENE EXPRESSION BY ANTISENSE RNAs (i) Transcriptional termination - Base pairing can mimic a terminator hairpin leading to a premature termination of transcription (attenuation) (ii) RNA stability - dsRNA targeted by ribonucleases leading to a degradation of the mRNA (iii) Translation control - Binding of antisense RNA to mRNA to block Shine-Delgarno sequence (ribosome binding site) or start codon REGULATION OF TRANSLATION BY RIBOSWITCHES (i) Effector molecule PRESENT - Binding of ligand to RNA makes it fold into an alternative structure in which the Shine-Delgarno sequence is occluded - No translatin (ii) Effector molecule is ABSENT - Folding of RNA leaves Shine-Delgarno sequence free for ribosomes to bind - Translation
How is the mal regulon regulated?
REGULATION OF MAL OPERONS - The inducer (maltose) binds to MalT and induces an allosteric transition allowing MalT to bind to the MalT box -> promotes RNAP binding and initiation of transcription - In the absence of maltose, MalT cannot bind and transcription is not initiated - Subject to catabolite repression - Regulated by two activator proteins CATABOLITE REPRESSION - CRP-cAMP complex activates expression of MalT - MalT can then activate expression of mal operons (along with CRP-cAMP)
How do certain antibiotics affect bacterial transcription? What mechanisms can bacteria develop for resistance?
RIFAMYCIN/RIFAMPIN/RIFAMPICIN - Inhibits transcription initiation by binding to the β subunit and blocks RNA exit channel - RNAP can only add a few nucleotide but then transcription ceases as RNA cannot exit the RNAP - Used for treating Mycobacterium infections - Mutations in the gene encoding β subunit (rpoB) can result in resistance due to lower binding affinity ACTINOMYCIN - Inhibits elongation - Consists of two cyclic peptides and flat ring (base analog) which mimics a nucleotide and intercalates between DNA G:C base pairs, immobilising the complex
Name the genes and proteins encoded in the lac operon.
Regulatory gene: lacI (located immediately upstream) Structural genes: (i) lacZ: codes for beta-galactosidase - Breaks down lactose into allolactose (an isomer of lactose), as well as glucose and galactose - Can also break down beta-galactoside (e.g. ONPG) to produce a colour reaction (ii) lacY: codes for permease - Membrane bound protein - Transports beta-galactosidase into the cell (iii) lacA: codes for thioglactosidase transacetylase - Function not completely understood; not essential for lactose fermentation - Transfers acetyl groups to beta-galactosidase to detoxify potentially harmful by-products
What is the trp operon? (of E. coli)
Required for synthesis of amino acid tryptophan - Anabolic (biosynthetic) pathway - Often regulated via a repressible system PRESENCE of tryptophan will REPRESS the trp operon - Only needs to be expressed when tryptophan level are low
What does attenuation require? Where does it occur?
Requires that transcription and translation occur at the same time (i.e. are coupled) so that transcription and translation can be terminated Does not occur in eukaryotes but in bacteria and archaea
Define the function of a restriction enzyme and a DNA probe.
Restriction enzymes recognise specific DNA sequences and cute DNA at those sites - Widespread among prokaryotes - Recognise palindromes (IR sequences) - Sticky and blunt ends - Protect from cell invasion by foreign DNA
Why should gene expression be regulated?
Specific genes are should only be active when required To allow control over the level of protein So energy is not wasted
What is the MalT DNA binding site?
THE MalT BOX - Most mal genes lack a -35 sequence in their promoters and instead have this - 5' GGGGA(T/G)GAGG 3' - Often found in direct repeats upstream of the transcription initiation site
Differentiate between transcription and translation initiation sites.
TRANSCRIPTION The promoter sequence is recognised by sigma (σ) factors - guide the RNA polymerase to promoters to initiate transcription. Operon organisation of bacterial genes results in the synthesis of polycistronic transcripts (multiple coding sequences on the same RNA molecule). Polycistronic transcripts containing rRNA and tRNA need to be cleaved/'processed' into individual units to create functional rRNAs and tRNAs TRANSLATION Sequences in the mRNA upstream of the start codon (AUG - towards the 5' end) is recognised. Consists the recruitment of ribosomes.
Define and describe transposons and their mechanism of transposition.
TRANSPOSON: mobile genetic elements which can move anywhere in a genome by two mechanisms "copy and paste", "cut and paste" TRANSPOSITION - Mediated when transposase recognises IR sequences - Transposase recognises target sequence -> cuts in staggered fashion - Duplication of target sequence MECHANISM OF TRANSPOSITION (i) Conservative - Transposon is cut out of original host and moves to target sit - Original DNA left with a break (ii) Replicative - Transposon is replicated and the second copy moves to the target DNA
Explain how sanger DNA sequencing technology works and provide examples of when such technology would be used.
Target DNA is copied many times, making fragments of different lengths. Fluorescent "chain terminator" nucleotides mark the ends of the fragments and allow the sequence to be determined. INGREDIENTS - DNA template - DNA primer - DNA polymerase - dNTPs: deoxyribonucleotide - ddNTPs: dideoxyribonucleotide METHOD - 4 separate reactions of different lengths (i) Incubation of ssDNA with unknown sequence inn DNA synthesis reaction mixtures containing ddNTPs (ii) Products of the reactions created (iii) Electrophoresis of reaction mixtures - number of nucleotides in fragment (iv) Autoradiography to visualise bands and deduction of 5' -> 3' sequence of newly synthesised DNA strand by reading order of bands from bottom to top USES AND LIMITATIONS - High quality sequences for long stretches of DNA - But expensive and inefficient for large-scale projects
Describe the basic features of gene expression. Define transcription and translation.
The central dogma of molecular biology: DNA (transcription) RNA (translation) Protein Transcription: the process by which RNA is synthesized using only one DNA strand as the template Translation: decoding of mRNA to make the protein encoded by the gene REFER TO NOTES FOR STEPS
What is the function of lacI in partial diploids?
The lacI repressor protein can bind to any functional lacO sequence - Binds to the operator of the lac operon on the main csome AND the F' plasmid lac operon THE lacI GENE PRODUCT AN ACT IN CIS AND TRANS Cis = regulates the expression of genes located on the SAME DNA molecule from WHICH IT IS ENCODED OR DERIVED Trans = regulates the expression of genes located on DIFFERENT DNA molecule from which it is NOT ENCODED OR DERIVED
Define gene expression.
Transcription of DNA into RNA, which is subsequently translated to make protein.
Define horizontal gene transfer and vertical gene transfer.
VERTICAL GENE TRANSFER Genes are passed onto offspring HORIZONTAL GENE TRANSFER Three mechanisms: (i) Conjugation - direct cell-to-cell contact via sex pilus (ii) Transformation - uptake of naked DNA (iii) Transduction - transfer of DNA via a bacteriophage DNA elements that are involved: - Plasmids - Bacteriophages - Transposons - Genomic/pathogenicity islands
What are the binding site for the AraC regulator?
araI1, araI2, araO1, araO2 Binding of AraC to: - araI1 + araI2 = +ve regulator, expression of ara genes - araI1 + araO2 = -ve regulator, no expression of ara genes Under catabolite repression mediated by CRP
How do mutations affect expression of the lac operon?
lacI - lacI^- = deletion of gene or mutation of DNA binding domain such that the repressor can no longer bind to lacO - lacI^s = super repressed; mutation in the effecto binding site which means allolactose cannot bind; lacI remains permanently bound to lacO lacO - lacO^c = mutation in the operator sequence - lacI cannot recognise the operator - No repressor binding - Constitutive expression of the lac operon
How do mutations affect lactose fermentation?
lacZ - No enzyme activity, no catabolism of lactose lacY - Depending on the type of mutation - Inability to take up or reduce lactose uptake ability lacA - No effect on ability to metabolise lactose - Other consequences not known
Explain replicative transposons (Tn3) and its mechanism.
~5,000 bp Contains three genes: tnpA, tnpR, bla 38 bp inverted repeats (IR) Transposition module: - tnpA: transposase - tnpR: resolvase - res site Beta-lactamase resistance MECHANISM (i) Catalysed by tranposase - Staggered cut at IR and target molecule - Cointergrate is formed between two DNA molecules (ii) Resolution - Cross-over occurs at res site - TnpR dimers bind to res - When a tetramer is formed with two res sites, cleavage occurs