Genet 270 lec 2- bac chromo
What did Griffith, and Hershey & Chase (1950s) demonstrate?
That DNA is the genetic material.
What is Chargaff’s Rule?
%G = %C and %A = %T.
What did Franklin & Wilkins discover in the 1950s?
DNA is a double helix.
What did Watson & Crick do in 1953?
Proposed the DNA double helix model.
What organism did Griffith use in his “Transforming Principle” experiment? (1928)
Streptococcus pneumoniae.
Which strain is virulent: Smooth (S) or Rough (R)? Why?
Smooth (S) strain; it has a polysaccharide capsule that protects from immune attack.
What was Griffith’s key finding?
Heat-killed S + live R → mice died; R bacteria transformed into virulent S by a “transforming principle.”
What did Avery, MacLeod & McCarty identify as the “transforming principle”? (1944)
DNA.
How did they prove it?
DNA fraction could confer s phenotype on r bacteria; transformation unaffected by protease or RNase, but destroyed by DNase.
What was the Hershey & Chase experiment about?
Showed DNA, not protein, carries genetic information using T2 phages.
What labels did they use for DNA and protein?
DNA → ³²P, Protein → ³⁵S.
What was the hershey chase experiemtn and result?
infect bac with labelled t2 phages, agitate bac in blender to seperate phage coats from bac. 70% ³²P entered bacteria and was passed to progeny phage; 80% ³⁵S stayed outside in phage coats. progeny phages had majority (30%- 32P) label.
what did the hershey chase expeirment suggest
that heredity is similar both in bacteria (prokaryotes) and higher organisms (eukaryotes)- both contain DNA as genetic material
What are the building blocks of DNA?
Deoxynucleotides (base + sugar + phosphate).
How are nucleotides linked?
Phosphodiester bonds (5′ carbon to 3′ carbon).
What are the ends of a DNA strand?
5′ phosphate and 3′ hydroxyl.
How are DNA strands oriented?
Antiparallel.
What stabilizes base pairing? whats more stable dna or rna
Hydrogen bonds between complementary bases. DNA is more stable than RNA, DNA lacks the 2′-OH group on its sugar, making it less prone to hydrolysis.
which bases are purine (double ring) and which are pyrimidine?
purines: Adenine Guanine
pyrmidine: Cytosine Uracil Thymine (CUT)
Why are G≡C base pairs stronger than A=T pairs?
G≡C pairs have 3 hydrogen bonds vs 2 in A=T, plus stronger base stacking interactions, making DNA with high G≡C content more stable. Higher G≡C content → higher melting temperature → more stability.
What is the nucleoid?
A mass of DNA in bacteria representing ~1000× condensation of the chromosome.
How is bacterial DNA arranged in the nucleoid?
~400 loops, each ~10 kb, connected to a central core.
Where is the nucleoid located in the bacterial cell?
Its position is carefully coordinated within the cell.
What processes influence nucleoid structure?
DNA replication, recombination, and transcription.
Is the nucleoid static or dynamic?
Dynamic — its shape and organization change with cell activities.
What role do NAPs play in the nucleoid?
Organize DNA and regulate gene expression.
How do NAPs shape DNA?
They bend, wrap, and bridge DNA.
How do different NAP binding modes affect the cell?
They alter nucleoid shape and regulate gene expression.
What is DNA supercoiling?
Bending of DNA back on itself due to under- or over-winding of the double helix.
What is positive supercoiling?
DNA is over-wound (strands wrap around each other more than relaxed DNA).
What is negative supercoiling?
DNA is under-wound (strands wrap around each other less than relaxed DNA).
How do NAPs influence supercoiling?
constrain super coils to prevent twisting. changes in nap binding can lead to unconstrained super coils, aids strand separation in replication, recombination, transcription.
what does toposimerase do vs dna gyrase
Topoisomerase I (Type I): Cuts one strand of DNA.Relieves negative supercoiling (relaxes underwound DNA).
DNA gyrase introduces negative supercoils (not positive) and helps remove positive supercoils ahead of replication/transcription.
How do RNA & DNA polymerases affect supercoiling?
They create positive supercoils ahead of replication/transcription forks.
What are topoisomerases?
Enzymes that modulate supercoiling by cutting, passing, and resealing DNA strands.
What are the two types of topoisomerases?
Type I: Cuts one strand, passes the other through, reseals; removes negative supercoils (TopA). ends as an infinity.
Type II: Cuts both strands, passes two strands through (from same or different DNA), reseals; includes Topo IV (decatenates chromosomes) and Gyrase (introduces negative supercoils). ends as an oval.
How big are bacterial genomes?
Range 0.5 Mb (~500 genes) to 10 Mb (~10,000 genes).
How does bacterial DNA compare to eukaryotic DNA?
Fewer introns, less repetitive DNA, densely packed (~1 gene/1 kb).
What types of genes do bacterial genomes encode?
Proteins, rRNAs, tRNAs, sRNAs, and small peptides.
What is synteny?
Conservation of genetic linkage/order across bacterial genomes.
Why is synteny important?
Indicates evolutionary conservation of genome structure.. bac genomes have high degree of synteny.
How can synteny be disrupted?
By insertions of DNA acquired through horizontal gene transfer. (DNA from different sources not ancestors)
What kinds of DNA elements are commonly acquired via HGT?
Prophages, insertion sequences, genetic islands.
How does E. coli O157:H7 differ from E. coli K-12?
O157:H7 has an extra 1 Mb of DNA (toxins & virulence factors) from HGT → pathogenic.
In which direction does DNA synthesis always occur?
5′ → 3′.
What is required to initiate DNA synthesis?
A 3′ OH group from a primer.
Which DNA polymerases are key in bacteria?
Pol III: large complex; Main enzyme for replication.
Pol I: Replaces RNA primers with DNA- DNA repair.
What does primase (DnaG) do?
Synthesizes short RNA primers for replication.
What do nucleases do?
Break down DNA. Endonucleases cut inside strands, exonucleases cut at ends (5′- pol 1 primer removal or 3′- editing).
What does ligase do?
Seals nicks in DNA by making phosphodiester bonds between 5′ phosphate and 3′ OH.
What accessory proteins assist replication?
Helicase: unwinds DNA.
Topoisomerase: relieves supercoiling.
Sliding clamp: holds Pol on template.
Clamp loader: loads clamp, binds pol on both strands and helicase
quick 6 steps of dna replicaton
1. pol 3 replicates DNA on leading strand, primase synthesizes primer in opposite direction on lagging
2. pol 3 extends rna primer (Okazaki fragment)
3. primase synthesizes another primer
4. pol 3 extends this primer till it touches previous primer
5. pol 1 removes rna primer and replaces it with dna
6. ligase seals nick
How is DNA replication semi-discontinuous?
Leading strand: continuous synthesis by Pol III.
Lagging strand: synthesized in short Okazaki fragments.
How are Okazaki fragments processed?
Pol I removes RNA primer → fills gap with DNA → ligase seals nick.
What does helicase (DnaB) do?
forms ring that Unwinds dsDNA using ATP- lots of energy; requires DnaC to load onto ssDNA.
What do single-stranded binding proteins (SSBs) do?
Coat unwound DNA to prevent reannealing.
How are leading & lagging strand synthesis coordinated?
Pols on both strands interact with sliding clamp (DnaN).
Connected by τ protein (also binds DNAB helicase).
Helicase interacts with primase.
DNA template on lagging strand is looped around
Why is this coordination important?
Ensures polymerization & unwinding occur at the same speed on both strands.
What is the trombone model of DNA replication?
Lagging strand loops out as Okazaki fragments are synthesized, resembling a trombone slide.
How does Pol III handle Okazaki fragments?
Dissociates at completed fragment, leaves clamp, hops to next primer site, continues synthesis.
Do leading & lagging strand polymerases stay associated?
Yes, they remain connected during replication.
What are common blocks to DNA replication?
Broken DNA, damaged DNA, supercoiling, and proteins bound to DNA (like RNA polymerase).
How is replication stalled by damage on the lagging strand resolved?
Pol III releases at lesion, leaving DnaN clamp.
Pol III reinitiates synthesis at a new primer.
DNA gap repaired later by another mechanism.
(stalled lagging strand DNA polymerase recycled to new DNA primer)
How is replication stalled by damage on the leading strand resolved?
DnaG primase synthesizes a new primer on sites of stalled pol III on leading strand.
Pol III releases at lesion, reinitiates synthesis from new primer.
Clamp loader complex (3 τ subunits/proteins in it) may bind 3 pol III, so that one is in reserve to recontinue DNA synthesis on other side of lesion
Why can’t Pol III replicate across DNA lesions?
Its catalytic pocket checks base-pairing accuracy → highly accurate but unable to bypass damage.
Which polymerases can perform translesion synthesis?
Pol II, Pol IV, Pol V.
How are they different from Pol III?
They are less accurate and less processive, but can replicate across DNA lesions.
How is their access to the fork regulated?
By polymerase switching and DNA-damage–induced expression.
What is the biggest problem for replicating polymerases?
Collisions with transcription machinery (RNA polymerase).
What are the two types of replication–transcription conflicts?
Head-on conflicts (most detrimental).
Co-directional conflicts (less severe but still problematic).
what is a head on conflict for replicating polymerases ? how has the genome evolved from this?
t’s when DNAP and RNAP collide while moving in opposite directions. These conflicts are highly detrimental, causing replication stress due to positive supercoiling and fork stalling. By orienting most highly transcribed genes co-directionally with replication forks, especially rRNA & tRNA genes, and by evolving accessory proteins that remove stalled RNAPs.
What is a co-directional conflict in DNA replication?
When DNAP and RNAP move in the same direction, causing DNAP to catch up and collide with RNAP. Less harmful than head-on conflicts but still problematic at highly transcribed genes. detrimental when multiple RNA pol involved (ex. at rrn operons)
Why are collisions likely between replication and transcription? Compare speeds of DNAP vs RNAP:
Replication fork moves much faster than RNA polymerase. DNAP III = ~1000 nts/sec; RNAP = 30–90 nts/sec.
How does bacterial genome organization reduce conflicts?
Highly transcribed genes (like rRNA & tRNA) are oriented co-directionally with replication.
Prevents lethal head-on collisions.
Why is bacterial genome organization important?
Minimizes transcription-replication clashes at essential, highly expressed genes.
What is the role of proteins like Mfd?
Dissociate stalled RNAP from DNA → reduce replication barriers.
How does Mfd assist repair? When do Mfd levels increase?
Recruits UvrAB repair proteins. In response to stress.
what does Mfd stand for
mutation frequency decline
Which proteins modulate RNAP activity during replication stress?
ppGpp, DksA, GreA, GreB.
How do they help? (proteins that modules RNAP activity)
Dislodge RNAP complexes stalled at lesions or during normal transcription → prevent replication-transcription conflicts. also can dislodge elongating RNAP in efforts of prevention of conflict in absence of DNA lesions (during normal growth) .
How do DksA, GreA, GreB act mechanistically?
Insert coiled-coil domains into RNAP’s secondary channel to promote dislodging.
Which accessory helicases assist replication through obstacles?
Rep, UvrD, DinG.
What is their function?
Remove stalled RNAPs or other DNA-bound proteins → prevent head-on collisions. help rep fork get through genomic regions where DNA binding proteins are found or recA coated ssDNA is found.
Can the replisome itself dislodge RNAP?
Yes — Pol III can knock RNAP off DNA. occurs in co directional conflicts
How is replication restarted afterward?
The RNA transcript can serve as a primer for DNA synthesis restart. occurs on leading strand.
What 3 major updates have been discovered about bacterial DNA replication?
A) DnaG primase can act on the leading strand → leading strand synthesis can be discontinuous (in vivo evidence).
B) Pol III may use RNA transcript as primer after co-directional collision to restart dna synth (in vitro evidence).
C) Replisome can switch Pol III for another Pol (II, IV, V) or another DNA pol II when needed (in vitro & in vivo evidence).