Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Can’t rely on glycosylation, disulphide bonds, lipidation, selective proteolysis, etc for function! Best bets are bacterial proteins Alternatives are eukaryotic proteins that don’t need any of the above Short peptides Tweaking p18 Linker Deleting or replacing GFP TRZN Oxalate decarboxylases Lactate dehydrogenase or other oxalate metab enzyme Something that may turn into thesis project!

Magnetospirillum gryphiswaldense Can propagate plasmids (but pBAM requires pir gene) Or can insert into chromosome via tnpA (Tn5)-based transposition

Magnetospirillum gryphiswaldense Can propagate plasmids (but pBAM requires pir gene) Can insert into genome by transposition no variation in expression due to copy # or growth stage

DNA replication Replication begins at origins of replication DNA polymerases are dumb! other proteins tell where to start

DNA replication Replication begins at origins of replication DNA polymerases are dumb! other proteins tell where to start bind origins & position DNA polymerase at start

DNA replication bind origins & position DNA polymerase at start Prokaryotes have one origin/DNA molecule copies of DnaA bind E. coli chromosomal ori C (cyan boxes). This unwinds adjacent DNA, allows DnaC to load helicase (DnaB) on yellow boxes to start assembling replisome

DNA replication copies of DnaA bind E. coli chromosomal ori C (cyan boxes). This unwinds adjacent DNA, allows DnaC to load helicase (DnaB) on 13 bp repeats

Prokaryotes have one origin/DNA molecule 1 on chromosome and one on each plasmid Plasmid numbers vary depending upon origin sequence i.e. factors that bind to them

Prokaryotes have one origin/DNA molecule 1 on chromosome and one on each plasmid Plasmid numbers vary depending upon origin i.e. factors that bind to them i.e. sequences and structure vary

Plasmid numbers vary depending upon origin i.e. factors that bind to them Plasmids in same “incompatibility group” use same factors, so should not be cotransformed

Plasmid numbers vary depending upon origin Plasmids in same “incompatibility group” use same factors, so should not be cotransformed Some, eg colE1 are controlled by antisense RNA

Others (eg R6K) are controlled by iterons: binding sites for regulatory proteins (eg Pi or RepA) DNA pol III needs primer RNA & DnaA to replicate

Many plasmids (pBS, pR6K) replicate by” theta mode”

SS-phage and some plasmids replicate by “rolling circle”

Other replicate by ”strand displacement”

Initiating DNA replication Prokaryotes have one origin/DNA molecule Pulse-chase experiments show eukaryotes have multiple origins/chromosome

Initiating DNA replication In Euk ORC (Origin Recognition Complex) binds ARS A is invariant, but B1, B2 and B3 vary A B1B2B3

Initiating DNA replication In Euk ORC binds ARS licensing factors ensure each ARS is only replicated once/S

Initiating DNA replication In Euk ORC binds ARS licensing factors ensure each ARS is only replicated once/S Activation factors initiate DNA replication

Initiating DNA replication licensing factors ensure each ARS is only replicated once/S Activation factors initiate DNA replication Licensing & activation factors fall off once replication starts, don't reattach until after mitosis

DNA replication must melt physiological T Helicase melts DNA

DNA replication must melt physiological T Helicase melts DNA Forms “replication bubble”

DNA replication Helicase melts DNA SSB proteins separate strands until they are copied

DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere

DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere DNA gyrase relieves supercoiling

Topoisomerases : enzymes that untie knots in DNA Type I nick backbone & unwind once as strand rotates Type II cut both strands: relieve two supercoils/rxn

DNA replication 1) where to begin? 2) “melting” 3) “priming” DNA polymerase can only add

DNA replication “priming” DNA polymerase can only add primase makes short RNA primers

DNA replication DNA polymerase can only add primase makes short RNA primers DNA polymerase adds to primer

DNA replication DNA polymerase can only add primase makes short RNA primers DNA polymerase adds to primer later replace primers with DNA

DNA replication 1) where to begin? 2) “melting” 3) “priming” 4) DNA replication

DNA replication add bases bonding 5’ P to 3’ growing end

DNA replication add bases bonding 5’ P to 3’ growing end Template holds next base until make bond

DNA replication add bases bonding 5’ P to 3’ growing end Template holds next base until make bond - only correct base fits

DNA replication add bases bonding 5’ P to 3’ growing end Template holds next base until make bond - only correct base fits - energy comes from 2 PO 4

DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off

DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off Proof-reading: only correct DNA can exit

DNA replication Proof-reading: only correct DNA can exit Remove bad bases & try again

DNA replication Only make DNA 5’ -> 3’

Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways!

Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways! Make leading strand continuously

Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way

Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments

Leading and Lagging Strands Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments each Okazaki fragment has its own primer: made discontinuously

Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous

Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous Okazaki fragments grow until hit one in front

RNAse H removes primer & gap is filled

Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments

Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments Energy comes from ATP-> AMP

DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome

DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome Attached to membrane & feed DNA through it

DNA replication Proteins replicating both strands are in replisome Attached to membrane & feed DNA through it lagging strand loops out so make both strands in same direction

DNA pol detaches when hits previous primer, reattaches at next primer

Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage

Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline

Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to C-terminus of mamC

Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface

Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface Purify with magnets

Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein

Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein Replace eGFP of pJH3 with your protein Best to use MluI and NheI sites

Assignment Best to use MluI and NheI sites Design oligos that add MluI in frame at 5’ end and NheI at 3’end

Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate

Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate  Find & analyze clones

Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )

Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )  not absolutely needed, but cells lacking  are very sick

Initiating transcription in Prokaryotes 1) Core RNA polymerase is promiscuous

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters Different sigmas bind different promoters

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters 3) Once bound, RNA polymerase “melts” the DNA

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template 6) sigma falls off after ~10 bases are added

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by  factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor Other sequences also often influence transcription! Eg Trp operator

Prok gene regulation 5 genes (trp operon) encode trp enzymes

Prok gene regulation Copy genes when no trp Repressor stops operon if [trp]

Prok gene regulation Repressor stops operon if [trp] trp allosterically regulates repressor can't bind operator until 2 trp bind

lac operon Some operons use combined “on” & “off” switches E.g. E. coli lac operon Encodes enzymes to use lactose lac Z =  -galactosidase lac Y= lactose permease lac A = transacetylase

lac operon Make these enzymes only if: 1) - glucose

lac operon Make these enzymes only if: 1) - glucose 2) + lactose

lac operon Regulated by 2 proteins 1) CAP protein : senses [glucose]

lac operon Regulated by 2 proteins 1)CAP protein : senses [glucose] 2)lac repressor: senses [lactose]

lac operon Regulated by 2 proteins 1)CAP protein : senses [glucose] 2)lac repressor: senses [lactose] encoded by lac i gene Always on

lac operon 2 proteins = 2 binding sites 1) CAP site: promoter isn’t active until CAP binds

lac operon 2 proteins = 2 binding sites 1)CAP site: promoter isn’t active until CAP binds 2)Operator: repressor blocks transcription

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation 2) Repressor blocks transcription if no lactose

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose 2) Repressor blocks transcription if no lactose 3) Result: only make enzymes for using lactose if lactose is present and glucose is not

Result [  -galactosidase] rapidly rises if no glucose & lactose is present W/in 10 minutes is 6% of total protein!

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. ↵

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator

Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR

Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR nrsR binds Ni promoter and activates transcription of both operons

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho”

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho” 3) rrnB first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription

Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Now have Pols IV & V in plants Make siRNA

Transcription in Eukaryotes RNA polymerase I: 13 subunits ( unique) acts exclusively in nucleolus to make 45S-rRNA precursor

Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis

Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis insensitive to  -aminitin

Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to  -aminitin Mg 2+ cofactor initiation frequency

RNA polymerase I promoter is 5' to "coding sequence" 2 elements 1) essential core includes transcription start site UCE -100 core +1 coding sequence

RNA polymerase I promoter is 5' to "coding sequence" 2 elements 1) essential core includes transcription start site 2) UCE (Upstream Control Element) at ~ -100 stimulates transcription x UCE -100 core +1 coding sequence

Initiation of transcription by Pol I Order of events was determined by in vitro reconstitution 1) UBF (upstream binding factor) binds UCE and core element UBF is a transcription factor: DNA-binding proteins which recruit polymerases and tell them where to begin

I nitiation of transcription by Pol I 1) UBF binds UCE and core element 2) SL1 (selectivity factor 1) binds UBF (not DNA) SL1 is a coactivator proteins which bind transcription factors and stimulate transcription