Chapter 7 Flashcards
Most bacteria and archaea have circular chromosomes
Some have linear or a mix of circular and linear chromosomes
Genomes vary in size
• Bacteria and archaea ~130 to 14,000 kilobase pairs (kb)
• Eukaryotic: 2,900 kb (Microsporidia) to 100,000,000 kb
(flowering plants); 3,000,000 kb (human)
Bacteria and archaea have very little noncoding DNA
DNA that does not encode proteins
Typically less than 15%
• Many eukaryotes have large amounts of noncoding
DNA (humans: over 90%)
gene
info composed of a sequence of DNA nucleotides
monocistronic RNA
A gene can operate independently of other genes
operon
gene 1 gene 2 gene 3 instead of just one gene.
• Creates a polycistronic RNA
promotor
DNA control sequence that launch RNA synthesis
regulon
genes and operons at diff positions in the chromosome that have a unified biochemical purpose
DNA function depends on its chemical
structure
• DNA is made of 4 different nucleotides linked by phosphodiester
purines
A, G (bycyclic, double ringed form)
Pyrimidines
T, C, U (monocyclic, single ringed form)
hydrogen bonding between bases
- A – T
* G – C
dna double helix grooves
- Major groove
- Minor groove
- Provide DNA-binding proteins access to nucleotides
DNA must be compacted to
fit into cell-into the nucleoid
histone like proteins
DNA ends must be tethered to form supercoils
• Supercoils are introduced by
- Cleaving both strands at one site
- Passing intact part of molecule b/w end of the cut site
- Reconnecting the free ends
Nucleoids of bacteria and most archaea are
negatively supercoiled (underwound) – easier to separate during transcription
topoisomerases
Enzymes that change DNA supercoiling
topoisomerases type I
single proteins that cleave one strand of a double helix
• unwind/relieve supercoils
topoisomerases type II
multiple subunits that cleave both strands of DNA molecule
• Introduce negative supercoils
• DNA gyrase
DNA gyrase complex (example of Topoi. II) is a
tetramer composed of two GyrA and two GyrB subunits
• GyrA is ATP-dependent
Quinolone antibiotics
target type II topoisomerases (do not affect eukaryotic topoisomerases)
• Stabilize the complex in which DNA gyrase is covalently attached to DNA
• Creates a physical barrier that blocks DNA replication
DNA replication is divided into 3 phases
initiation, elongation, termination
DNA REPLICATION initiation
unwinding of helix and loading DNA polymerase
DNA REPLICATION elongation
addition of deoxyribonucleotides to growing DNA chain, followed by proofreading
DNA REPLICATION termination
the DNA duplex is duplicated, the negative supercoils are restored, and key sequences of new DNA are methylated
replication begins at
origin (oriC) (245-bp), replicates bidirectionally.
In E. coli, initiation is activated by
inhibited by
- protein DnaA
- SeqA
DNA methylation controls timing of SeqA binding
IN E COLI INITIATION
• Deoxyadenosine methylase (Dam) attaches CH3 to N-6 position of A in sequence GATC
• lag b/w synthesis of new DNA and methylation
• Origin is temporarily hemimethylated (only one strand is methylated)
• SeqA has high affinity for hemimethylated origin sequence
• Binding of SeqA prevents another initiation event until
sequence is fully methylated
Binding of DnaA to the origin facilitates melting and initiates formation of the replisome
5 DnaA bind in E. coli, 15 for B. subtilis
Another round of replication can only begin after
- Origin is fully methylated
- SeqA dissociates
- DnaA-ATP concentration rises
dna helicase
DnaB
Dna helicase holder
DnaC
(initiation) replisome
Two DNA polymerase III, DNA primase (DnaG), and
helicase (DnaB)
(elongation) E. coli contains 5 DNA polymerase proteins
Pol I to Pol V
• 5’-to-3’ direction
• Only Pol III and Pol I participate directly
(elongation) DNA Pol III
- Alpha subunit – DNA synthesis
- Epsilon subunit – DnaQ – proofreading
- Other subunits also involved in improving accuracy
(elongation) leading strand
synthesized continuously
(elongation) lagging strand
– synthesized discontinuously in pieces (Okazaki fragments)
(elongation) single-stranded DNA-binding proteins (SSBs)
approx every
during lagging strand ssDNA is protected from degradation
• approx. every 1000 bases new RNA primers are
synthesized by DnaG
(elongation) Lagging strand left with patches
of RNA primers
(elongation) DNA Pol I 5’-to-3’ exonuclease activity or
RNaseH
cleaves RNA primers
• RNaseH recognizes RNA-DNA hybrids
uses 3’-OH end as a primer to fill in the gap
• uses dna ligase
(elongation) DNA ligase with energy from NAD
forms the phosphodiester bond
(termination) A series of terminator sequences
stops replication
- 10 ter sequences in E. coli
* One set stops the CW replicating polymerases, one set for the CCW.
Tus – terminus utilization substance
binds to ter sequences and stops DnaB helicase activity
(termination) catenane
Replicated chromosomes appear as linked rings
• XerC and XerD cut and rejoin
plasmids
found? usually need can contribute to \_\_\_\_ bw cells
• Plasmids are extragenomic DNA molecules
-Smaller than chromosomes
• Found in archaea, bacteria, and eukaryotic microbes
• Usually circular
-Typically negatively supercoiled
• Need host proteins to replicate
-Replication not tied to chromosome replication
• Can contribute to the physiology of the cell
-Antibiotic resistance
• Transmitted between cells
-Conjugation
two replication methods IN PLASMIDS- bidirectional and
- RepA-
- RepA holds
- RepA recruits
• Rolling-circle – unidirectional
- RepA-replication initiator binds origin and nicks 1 strand
- RepA holds onto 5’-phosphate of nicked strand while 3’-OH is primer for DNA polymerase
- RepA recruits helicase to unwind DNA and SSBs bind
- Nicked strand is rejoined by RepA
In some cases it is just by chance
that the daughter cell inherits a plasmid.
high-copy plasmids
(50-700 copies per cell) have a high probability that each daughter cell will have at least one plasmid
low-copy plasmids
- Ex plasmid R1 in Salmonella uses genes parC, parM, and parR
- ParR-parC forms complex with the plasmid
- ParM protein is an actin-like filament – attaches to ParR-parC and pushes plasmid copies to opposite ends of cell
plasmids carry
genes beneficial to the host survival in a specific environment (e.g. antibiotic resistance)
restriction sites
Restriction endonucleases cleave unfamiliar DNA
- Palindromic sequences; 4-6 bases in length
- Cleave the phosphodiester backbones of opposite strands
- Create blunt or staggered (“sticky”) ends
Cloning
- Stanley Cohen and Herb Boyer
- “cut and paste” cloning
- Restriction cloning
Sanger sequencing
Uses
Specific
• Uses a DNA synthesis w/ ddNTP (stops elongation)
• Specific ratio of dideoxy- to normal deoxynucleotides = elongation stopping at diff points in DNA sequence
-Creates different lengths of DNA
Sanger sequencing –(number of bp)
1,000 bp in a few hours
Sequencing by synthesis
600 gigabases (Gb) per run • 1 Gb = 1 billion bases (Human genome ~3 Gb) • Depending on Illumina system and settings, one run can take 1 day – 1 week
enhancer
drives transcription in eukaryotic promoters. function at large distances from gene.
extreme thermophiles- reverse dna gyrase
introduces positive supercoils.
