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Bio3124 Lecture 8. Bacterial Genetics: I. Genome replication and packing. DNA Contains Cell Information. Total cell DNA = genome ( chromosome & extra-chromosomal ) Human genome = 4 billion bp 1000x as large as E. coli genome 90% junk DNA - PowerPoint PPT Presentation
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Bio3124Lecture 8
• Total cell DNA = genome (chromosome & extra-chromosomal)
• Human genome = 4 billion bp– 1000x as large as E. coli genome
– 90% junk DNA
– ~8x more genes: 30,000 (human) vs. 4,000 (E. coli)
• Bacterial genomes = 0.6–9.4 Mbp
– Genome of bacteria usually circular• Seldom linear, segmented
DNA Contains Cell InformationDNA Contains Cell Information
Bacterial Genetic OrganizationBacterial Genetic Organization
• E. coli genome– regulatory
• promoter/operator, signal sequences
– coding sequences– Average 1000 bases per bacterial
gene– Organized on both strands– Operons and regulons– Monocistron vs Polycistron
organization– Overlapping genes => ribosomal
frameshifting
ATGCCCCAA---//---CCAAAATGAACGAAAATCTGTTCGCTTCAT
Met Pro Gln Pro Lys Trp Thr Lys Ile Cys Ser Leu His
Met Asn Glu Asn Leu Phe Ala Ser
Overlapping genesOverlapping genes
DNA is an antiparallel double helixDNA is an antiparallel double helix
• Geometry of bases and their spacial arrangement to form H-bond cause helix structure of dsDNA
• B-form DNA
• pairing bases stack at the centre
• backbone intertwined• creates minor and major grooves
• 0.34 nm (3.4 A) rise per base pair
• one full helix turn houses 10 nucleotides
34 A
Major groove
20 A
DNA is an antiparallel double helix
34 A
Major groove
20 A
DNA Is Packed to Fit the CellDNA Is Packed to Fit the Cell6
DNA Is Packed to Fit the CellDNA Is Packed to Fit the Cell
• Multiple loops held by anchoring proteins
• Each loop has coiled DNA
Nucleoid of E. coli Circle of dsDNA 1500x the
size of the cell
Supercoiling Compacts DNASupercoiling Compacts DNA
• Unsupercoiled DNA = 1 winding for 10 bases• Positive supercoils– Winding more frequently • Overwinding
• Negative supercoils– Winding less frequently• Underwinding
• Supercoils twist DNA• Why supercoils are important?– Eubacteria => less frequent winding– Extreme thermophiles => more frequent winding
CircularLinear
Super-coiled
Ladder
1 2 3
Relevance to ResearchRelevance to Research
Topoisomerases Regulate SupercoilsTopoisomerases Regulate Supercoils
• Type I Topoisomerases – Relieve torsional stress caused by
supercoils– Act on one strand, How?
• Type II Topoisomerases (DNA gyrase)– Unwind dsDNA– Introduce negative supercoils– Act on both strands of dsDNA, How?
• Archaeal topoisomerases– Reverse topoisomerases– Introduce positive supercoils
Topoisomerase ITopoisomerase I• Single protein, nicks one strand• Allows passages of the other strand through single strand
break• Releaves accumulated positive supercoils ahead of
replicating DNA
Topoisomerase II (DNA Gyrase)Topoisomerase II (DNA Gyrase)
• two subunits, GyrB and GyrA• GyrB binds DNA, passes to GyrA• GyrA introduces double strand break– 2 ATP hydrolysed– Remains transiently attached
• Passes other dsDNA through break• Reseals the ds break• A negative writhe introduced
Mechanochemical analysis of DNA gyrase
Topoisomerases Regulate SupercoilsTopoisomerases Regulate Supercoils
Summary Animation: Topoisomerases I and II Summary Animation: Topoisomerases I and II
DNA ReplicationDNA ReplicationSemiconservative replication• Copies information from one strand to a new,
complementary strand– Dividing cells receive one parental strand and one
newly synthesized strand
– Melt double-stranded DNA– Polymerize new strand complementary to each melted
single strand
oriC
ter
‘9-mers’ ‘13-mers’
Replication Begins at Replication Begins at oriCoriC
E. coli oriC: 245 bp
Replication Begins at Replication Begins at oriCoriC• Timing: Dam methylation at
A of GATC (ie. GAN6mTC)• SeqA binds to hemi
methylated duplex at OriC• Full methylation following
cell division and loss of SeqA affinity
• DnaA concentration rises• Binds to 9-mer repeats at
OriCOriC: 245 bp contining 9-mer repeats, with 13-mer repeats in betweenDnaA binding, strand melting at 13-mer by RNAP
DNA Helicase Melts DNA• Helicase Loader (DnaC) places helicase (DnaB) at
each end of origin
Helicase
Origin
Loader
Helicase Recruits PrimaseHelicase Recruits Primase
• Primase begins replication• RNA primer forms 3OH for DNA to attach– Evolutionary remnant?– 1st cells thought to use RNA, not DNA
Helicase
Primase
Primosome
Primer Recruits Clamp Loader to Each StrandPrimer Recruits Clamp Loader to Each Strand
• Sliding clamp binds DNA polymerase III to each strand
DNA Pol III
DNA Pol III
SlidingClamp
Clamp Loader
Polymerase Proceeds 5 3 on Each Strand
• Energy for polymerization comes from phosphate groups on added base.– Must add new base to 3OH of a chain– New nucleic acids grow to extend 3 end
Each Fork Has Two Strands
• Steady growth of new “leading” strand– Leading strand follows helicase
• Lagging strand: discontinuous, needs intermittent release and reloading of replisome
Leading Strand
LeadingStrand
Lagging Strand
Lagging Strand Growth• Polymerase continues to
previous primer• Clamp loader places
primase on new site• DNA present in 1000 base
pieces– Okazaki fragments
RNase H Removes Primers• One primer for each
leading strand• Many primers on
lagging strands– One per Okazaki
fragment
• Gaps filled in by DNA Polymerase I
• Ligase seals nicks
DNA Replication: Sliding modelDNA Replication: Sliding model• Replisome anchored to
membrane at mid-cell• DNA spools through as
replicated• Proof?• PolC-GFP stays at
equator attached to membrane
• DAPI stained DNA: throughout cytoplasm
Animation:Animation: Summary of DNA Replication Summary of DNA Replication
10 20 30 40
PCR cycles
• DNA replication in vitro• Polymerase chain reaction (PCR)
– Amplifies specific genes from a given genome– Need: template DNA, primers, dNTPs, DNA Polymerase,
buffer, Mg2+ fd– Denaturation, Annealing, Elongation
Relevance to ResearchRelevance to Research
Animation:Animation: Proof reading function of Pol III Proof reading function of Pol III
Both Forks Move to Both Forks Move to terter Sites Sites
• Movement is simultaneous• Opposite directions until both meet again at
terminus• Replisome disassembles at ter sites
PlasmidsPlasmids
• Extrachromosomal pieces of DNA• Low-copy-number plasmids– One or two copies per cell– Segregate similarly to chromosome
• High-copy-number plasmids – Up to 700 copies per cell– Divide continuously– Randomly segregate to daughter cells
Plasmid Genes
• Advantageous under special conditions – Antibiotic-resistance genes– Genes encoding resistance to toxic metals– Genes encoding proteins to metabolize rare food
sources– Virulence genes to allow pathogenesis– Genes to allow symbiosis
Relevance to ResearchRelevance to Research
• Molecular cloning– Plasmids are used
to import a segment of exogenous DNA into a host cell.
Plasmid ReplicationPlasmid Replication Bidirectional replication
Similar to chromosomal replication Unidirectional (“rolling circle”) replication
Starts at nick bound by RepA proteinProvides 3OH for replicationHelicase moves around
plasmid repeatedlyComplementary strand
synthesizedUsed by many bacteriophages
Animation: Rolling circle replication Animation: Rolling circle replication