BIOC0005 - Lecture notes ALL PDF

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Lecture 1 01.10 Chargaff’s rule: A=T G=C (three hydrogen bonds between C-G and 2 hydrogen bonds between T-Ah) Purine base pairs with a pyrimidine endonuclease : cleaves bonds internally exonuclease : start at one end and cleaves bonds one at a time Restriction endonuclease: cleaves ​both strand​s of...

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Lecture 1

01.10.2018

Chargaff’s rule: A=T G=C (three hydrogen bonds between C-G and 2 hydrogen bonds between T-Ah) Purine base pairs with a pyrimidine endonuclease : cleaves bonds internally exonuclease : start at one end and cleaves bonds one at a time Restriction endonuclease: cleaves b  oth strands of dsDNA at specific sequences. Cuts can be staggered recognition sites are palindromic EcoR1 = Escherichia coli R1(5’ overhangs AG cut) Pst1 = Pseudomonas stuartii 1 (3’ overhangs AG cut) Hpa1 = Haemophilus parainfluenzae 1 (blunt ends AT cut) DNA ligase (join DNA) - catalyse the formation of a phosphodiester link between the 5’ phosphate and 3’ OH of adjoining nucleotides in duplex. Hyperchromicity: Absorbance increases when ordered double stranded to disordered single stranded DNA DNA Tm depends on DNA composition Ethidium bromide intercalates between stacked bases in DNA, it fluoresces when exposed to UV light

Research Project

01.10.2018

Aim of the project ● To create a genomic library of bacteriophage lambda ● To isolate, from the library, a lambda DNA fragment ● To identify the cloned lambda fragment A library is a collection of bacterial colonies that each contain a different fragment of the target DNA 1. Set up the restriction digests 2. Once the DNA has been incubated a small sample will be checked by agarose gel electrophoresis to check the restriction digests. 3. Treat pUC19 molecules cut with alkaline phosphatase to remove 5’ phosphate groups

Plasmid ● ● ● ● ● ●

Prof. Santini

Extrachromosomal DNA molecule Circular or linear Autonomous replication Range in size from kilobases to megabases Control their copy number Ensure inheritance at each cell division by process called partitioning

04.10.2018

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Plasmids with same replication machinery cant co-exist in the same cell- known as incompatibility. Molecular properties of plasmids ● Compact conformation - supercoiled ● Small high copy - random plasmid partitioning ● Large low copy - 1-2 copies per cell, directed plasmid partitioning. - replication linked to chromosome replication. Plasmid incompatibility Several plasmids can co0 exist in the same cell but not all co-exist stably. Narrow-host-ranged can only replicate in related species Broad-host-ranged can replicate in many different species Some characteristics that plasmids encode ● Antibiotic resistance ● metal/metalloid resistance ● Virulence determinants - animal and plant pathogenicity ● Bacteriocin production ● Biodegradative capabilities ● Symbiotic determinants - ability to fix nitrogen

Virulence determinants (pWR501) ● pXO1 - anthrax toxin - AtxA ● pXO2 - capsule production - protection against immune system ● pXO1 loss = loss of virulence ● pXO2 used as live attenuated vaccine for some Bacillus strains Bacteriocin = antimicrobial agent used to kill bacteria not harbouring plasmid that confers immunity/resistance to the compound. Transposon ● DNA sequence with ability to move ● IR = inverted repeat ● Bla encodes β--lactamase which confers resistance to ampicillin Fitness cost of plasmids ● Plasmids confer energetic costs to hosts ● Plasmids that proved fitness benefits are stably maintained in the host ● Plasmid and strain can co-evolve to decrease overall fitness cost to the strain Requirement to be a good vector - oriV - Selectable marker - MCS(multiple cloning site)

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Promoter upstream of MCS

Suicide vector A plasmid which is used with an oriV that is unable to replicate in host of interest Shuttle vectors A plasmid that contains two origins of replication allowing for replication in two hosts

08.10.2018 ● ● ●

Bacteriophage Lambda

Linear ds DNA 48,514bp 12bp cohesive ends 46 genes identified

Phage life cycles - Lytic growth 1. Infection 2. Early development 3. Late development 4. lysis - Lysogenic growth 1. Infection 2. Phage DNA is integrated into bacterial genome. 3. Induction

TERMINOLOGY - virulent - temperate - Lysogen - Prophage - Immunity - Induction: event that cause lysogen to release Lysis/lysogeny - Depends on conditions - Lysogeny favoured by 1. Low levels of nutrients 2. High MOI - Plague appearance - clear or turbid Lytic development controlled by a cascade Early stage: phage genes are transcribed by host RNA polymerase regulator gene products:

RNA polymerase Sigma factor or Antitermination factor Middle stage: early product causes transcription of middle genes Regulator gene & structural gene products: Sigma factor or antitermination factor Replication enzymes Late stage: middle product causes transcription of late genes Structural gene product: phage components Key points ● The early genes are transcribed by host RNA polymerase includes regulators required for delayed early expression ● Delayed early expression includes regulators for transcription of the late genes ● Expression of groups of genes occurs in an ordered manner. Lytic cycle in detail Early: Promotor L & R Promotor L transcribes NmRNA(antiterminator) binds to DNA and deactivate the terminator. Promotor R transcribes cro mRNA Delayed Early: pN permits transcription from same promoters to continue past N and cro Late: transcription initiates at pR’  and pQ permits it to continue through all late genes The late genes form a single transcription unit Two methods exist for DNA replication 1. β(semi conservative) replication initiated by protein O & P, recruits the bacterial proteins 2. Rolling circle Lysogeny Lambda repressor encoded by cl is required to maintain lysogeny The repressor acts at OL and OR to block transcription of the immediate early genes This repression prevents the lytic cycle from proceeding; repressor dimer binds to lambda DNA prevents RNA polymerase from binding PL. Cleavage of monomers disturbs equilibrium, so dimers dissociate Lambda repressor - key points ● A repressor monomer has a N-terminal domain binds DNA and a C-terminal domain dimerises ● Binding to the operator requires the dimeric form ● Cleavage of the repressor reduces the affinity for the operator and induces a lytic cycle C1 determines whether bacteriophage undergo lysogenic stage or not Lysogeni - key point ● cII and cIII casue repressor synthesis to be established and trigger inhibition of late gene transcription ● Establishment of repressor turns off early gene expression

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Repressor maintains its own synthesis Lambda DNA is integrated in the final stage of establishing lysogeny

Lambda repressor - a 27k dalton protein Monomers are in equilibrium with dimers which bind to DNA, cleavage of monomers disturbs equilibrium so dimers dissociate Lambda insertion - key points ● Phage integrates into the chromosome by recombination between the attP site on the phage and attB on the bacterial DNA ● Excision occurs via recombination between sites at the end of the linear prophage ● Lambda int codes for an integrase that catalyses the intergration reaction. Xis is additionally required for excision. Operator region - key points ● Operator has three sites, each composed of two half sites ● Lambda repressor and cro each bind to these ● Repressor has highest affinity for OR 1 and oL 1 and co-operative binding to the second sites ● Cro has highest affinity for OL 3 and OR 3 Lytic cycle initiation- key points  and blocks the ● Wehn cro binds to OR 3 it prevents RNA apolymerase from binding to pRM maintenance of repressor protein. ● When cro binds to the other operator sites it prevents RNA polymerase from binding to PL and PR and turns off early gene expression. Lysogeny requires repressor to take over OL and OR Lytic cycle requires Cro to take over OL and OR. The critical event is whether cII causes sufficient synthesis of repressor to overcome the action of cro.

11.10.2018 understanding our genome Steps to learn about DNA ● Clone DNA ● Seuqence DNA ● Analyse sequence information Genomic DNA libraries - The library includes clones that cover the whole genome cDNA libraries - the library includes clones that correspond to the nRMA sequences Olia dT chromatography-is used to separate mRNA from the other RNAs in the cell

Cloning and sequencing the human genome. Synthesis of double-stranded cDNA ● mRNA is copied to cDNA (reverse transcriptase dNTPs, oligo dT primer) ● Phosphodiester bond cleavage(ribonuclease) ● RNA is replaced by DNA (DNA polymerase) ● DNA ligase -> double-stranded cDNA Human genome project - sanger centre BAC( baterial artiftial chromosome) ● BACs are used to create libraries with larger fragments ● BACs steps same as your research project ● BUT insert size has to be ~100,000bp ● This requires several special precautions Method 1. White blood cells are mixed with agarose and placed in a mould 2. Cell wall is ruptured 3. Restriction enzyme is added to digest DNA in the agarose mould 4. Each mould is placed in a well of a agarose gel 5. Gel is run, viewed under UV light and DNA of ~100,0 6. DNA is eluted from the excised agarose 7. Ligated to a plasmid vector cut with the same restriction enzyme 8. Treated with DNA ligase 9. Bacteria are transformed 10. Transformed bacteria are picked into 384-well plates 11. Bacterial DNA isolated for sequencing reactions Cloning and sequencing the human genome(contiguous, partial digestion with restriction enzyme) The first sequence of the human genome was deciphered using the dideoxy chain termination method Dideoxy chain termination sequencing of DNA Illumina method Mitochondrial genome contains 2 rRNA genes, 22 tRNA genes, 13 polypeptide coding genes(oxidative phosphorylation) H strand encodes 28 genes (G rich) L strand encodes 9 genes (C rich) No introns

Understanding our genome The life cycle of an L1 retrotransposon Most LINES are truncated at their 5’ end Suggest: reverse transcriptase does not fully transcribe the entire LINE mRNA Truncated LINES are not transcribed Insertion of LINES can cause gene disruption LINE transposition is rare in somatic cells more abundant in the germline Humans can differ in the number and position of their LINES Consequences of LINE insertion into the human genome Insertion into promoter - can silence a gene Insertion into intro - can slow down transcription SINES, short interspersed elements SINEs all have RNA pol III internal promoters Insertion of a SINE element into the genome ● SINE is transcribed by RNA pol III ● SINE mRNA is copied by reverse transcriptase provided by a LINE ● Process of integration thought to be similar to LINE integration Analyse microbial species by sequencing the microbiome- 16S rRNA gene sequencing

Method to sequence RNA Remove mRNA from rRNA and tRNA using oligodT chromatography. Fragment mRNA to ~50 to 100 ribonucleotides Convert RNA fragments into single strand DNA using random primer or oligodT and reverse transcriptase. Convert single stranded DNAs to double-stranded Add adaptors Prepare clone library and sequence all clones

Gene regulation in bacteria -

Transcription and its control

3 important factors for transcription 1. DNA-binding proteins 2. Specific DNA sequences

3. Environment- available nutrients Different subunits play different roles in transcription - RNA polymerase ● Recognise the beginning of a gene(BINDING) ● Insert correct nucleotide into position(INITIATION) ● Catalyse formation of phosphodiester bond(ELONGATION) ● Recognise the end of a gene(TERMINATION) The sigma subunit of RNA polymerase recognises and binds to a specific sequence called the promoter located at the beginning of the gene to be transcribed -35 region = recognition site -10 region = orientates RNA polymerase Other factors associate once sigma is bound consensus(most common) sequence of sigma70 promoters

Strong promoters - also contain an UP-element, RNA polymerase binds efficiently and transcription proceeds effectively The promoter weakness is needed to allow sophisticated nutrient regulation of the operon. Elongation ● Binding of correct nucleoside triphosphate ● Phosphodiester bond formation ● RNA polymerase moves one nucleotide at a time along DNA ● Melting and movement creates transcription bubble ● 5’ end of RNA chain is displaced as DNA helix reforms Termination ● RNA synthesis must end ● Newly synthesized RNA must be released ● RNA pol must dissociate from the DNA template 2 types of RNA synthesis termination 1. Rho independent(intrinsic terminators) GC rich region creates hairpin loop, Weak A:U bonds RNA chain is released, DNA helix reforms, RNA pol released 2. Rho dependent(requires the protein, rho) Rho is a hexamer protein - 6 identical monomer subunits.

Binds to transcripts only after the protein has been translated and the ribosome has left. Terminators are C-rich and ~40 nucleotides long. GENE EXPRESSION Control of gene expression ●

Genes coding for proteins involved in same metabolic pathway are clustered into an OPERON Operon is transcripted to one mRNA(polycistronic mRNA) Turn on(inducible)/off(repressible) transcription when needed/not needed Controlling with small molecule+regulatory protein Inducible operons Catabolic pathway Small molecule = substrate to first enzyme in the metabolic pathway. Active repressor for lac operon is a tetramer, 2 repressor molecules bound to each operator. Transcription of lac operon - negative regulation, operon is off when repressor is bound Positive control of lac operon ● Glucose inhibits lac operon transcription ● Glucose metabolism needs no new proteins

cAMP binds to CAP(catabolite activator protein)

cAMP-CAP binds to RNA pol to assist polymerase to bind with promoter, The promoter weakness allows the sophisticated nutrient regulation of the lac operon. cAMP-CAP binds with α CTD(carboxy-terminal domain) CAP bends DNA>90 degrees around the centre of symmetry This increases the affinity of RNA polymerase for lac promoter.

Two anomalies 1. Transport of inducer requires permease(lac y) 2. True inducer is allolactose NOT lactose

Binding of repressor never infinitely strong, drops off approx. once every cell generation, therefore: always low level lac transcription.

Repressible operons -transcription switched off when sufficient product is present in cell -

Tryptophan operon - trp operon

Tryptophan present - operon repressed Trp operon under negative control of the active repressor Attenuation(incomplete transcription) - also regulates trp transcription Attenuation depends on A. a folding pattern of leader mRNA

1 base pair 2 = no transcription 2 base pair 3 = transcription of operon 3 base pair 4 transcription only of leader mRNA B. extent to which ribosome has translated leader mRNA Transcription terminates when trp concentration is high Ribosome covers region 2, ribosome stops at leader peptide stop codon in region 2, therefore region 3 base pair 4, 3-4 stem-loop forms and terminates transcription. Attenuator stem loop - GC rich stem, U rich tail.

Two component regulatory systems Activator protein + regulator protein Phosphate - DNA, RNA, phospholipids, proteins Therefore transcription must be activated to increase [phosphate]

Active PhoB increases the affinity of RNA pol for promoter.

VAI increases transcription of the lux operons Lux R protein assists RNA pol to bind to promoter.

Bioluminescence

RNase P cut off the waste RNAs

25th October Bacterial conjugation

DEFINITION & HISTORY ● Transfer of genetic material between cells by direct cell-to-cell contact ● Discovered in 1946 by lederberg and tatum ● Plasmids or transposons can transfer from one organism to another by conjugation ● Occurs in the bacteria and archaea ● Most common mechanism of genetic exchange between prokaryotes ● Leads to acquisition of traits ● Important mechanism in evolution of prokaryotes Conjugative plasmids can encode: ● Antibiotic resistance ● Heavy metal resistance ● Pathogenicity islands ● Degradative capabilities ● Metabolic proteins Conjugative plasmids ● Two origins of replication ● oriV - vegetative replication ● oriT - origin of transfer - essential for conjugal transfer ● Conjugative or transfer genes establish a stable mating pair and trigger DNA transport from donor to recipient via a specialised transfer pore/channel Gram-negative systems ● F(fertility factor) is a narrow-host-range plasmid isolated from E. Coli ● RP4(Resistance factor - confers resistance to antibiotics) is a broad-host-range plasmid isolated from P. aeruginosa ● Ti(tumour inducing) plasmid is found in A. tumefaciens and is involved in causing crown gall disease in plants Nomenclature Conjugal transfer genes referred to as tra/trb genes when between prokaryotes And vir genes when between prokaryotes and eukaryotes. F plasmid

Transfers by conjugation and can also integrate into host chromosome by recombination and in doing so can also transfer host genes. Main steps: ● Mating-pair formation - pilus formation which is a type IV secretion system(T4SS) ● Signalling event that triggers DNA transfer ● DNA transfer which involves relaxosome formation ● Coupling protein - synchronises Mpf with Dtr and is thought to “pump” the DNA into recipient cell.

Mechanism of conjugation between two prokaryotic cells 1) Cell to cell contact made by pilus 2) Pilus retracts bringing cells closer together 3) DNA strand to be transferred is nicked at origin of transfer by relaxase 4) Relaxase also acts as a helicase unwinding DNA to be transferred 5) Rolling circle replication replaces DNA strand in the donor cell 6) Complimentary DNA strand is made in the recipient - recipient is now a donor. Sex Pili - 1-20μm long - structure has been solved

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8nm diameter Composed of identical 7.2kDa pilin subunits Pore 2 nm - enough space to accommodate single stranded DNA and protein

Relaxosome formation

Relaxosome = relaxase and accessory proteins Accessory proteins involved in relaxosome formation vary in different transfer systems but all contain the relaxase RP4 as the model ● Relaxase - TraI(essential) ● Accessory proteins - TraJ(essential) and TraH (stablise) ● Key enzyme is TraI which nicks one DNA strand and unwinds DNA = single stranded DNA molecule ready for transfer Stages of relaxosome formation 1. TraJ binds to inverted repeat closest to the site of DNA cleavage 2. traI binds to TraJ-oriT complex 3. Relaxosome formation is facilitated by the intrinsically bent region in oriT that allows in to wrap around a core of several subunits of Trak - enhances the fraction of plasmids that can be captured and cleaved at anic 4. traI-TraJ-oriT complex is stabilised by TraH Ti plasmid system - virB OPERON -virB1 - virB11 encodes proteins involved in Mpf - virD operon - virD1 - virD5 : virD1 and virD2 essential for Dtr and VirD4 is coupling protein(virD2 and virD1, equivalent of traI and ) T4SS - Type IV Secretion System ● Mediates translocation of macromolecules across cell envelopes of bacteria ● Conjugation Mpf system is a large subfamily of T4SS - translocation of DNA and proteins ● T4S can also function in the uptake of nucleic acids or proteins from the extracellular environment. Energetic components: VirD4, VirB4, VirB11 ● Large proteins>70kDa proteins with consensus motifs involved in nucleoside triphosphate(NTP) binding ● VirD4 - coupling protein, interacts directly with relaxosome and T4SS ● VirB4 - involved in energising the assembly or activity of the channel ● VirB11 - form as homohexamer rings and contain a central cavity = channel Inner membrane channel/scaffold proteins: VirB3, VirB6, VirB8 and VirB10 ● Proteins found in the inner membrane contributing to formation and activity of the channel ● VirB3 - Interacts with VirB4 and VirB2

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Involved in pilus assembly pathway and substrate translocation

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VirB6 - Interacts with DNA substrate mediating its transfer - Interacts with components of the channel