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-This section will be “gene centric” and concerned with:
what genes are
what genes do
how prokaryotes control gene expression
what we can learn by studying genes
-Goals:
1. Understand macromolecular synthesis
2. Understand gene regulation
3. Be able to apply our knowledge to ecology and viruses
What is a gene? DNA segment that encodes a protein or
RNA product (this def. needs to be expanded for virusesmore later)
-Meomorize this definition!!
Francis Crick laid out the relationship between DNA, DNA and
protein synthesis --this is now called the “Central Dogma”
Central Dogma
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DNA Structure
DNA=2-deoxyribonucleic acid
synthesized from dNTPs=deoxyribonucleotide triphosphates
-Precursors for nucleic acid synthesis are acquired from the environment (if
available), or synthesized de novo.
DeoxyRibose
purines and pyrimidines
Ribose: synthesized in the pentose phosphate cycle from C6, C3, C4 or C7
precursors.
Ribose ring structure
Used for precursors of RNA:
Deoxyribose ring structure
Used for precursors of DNA::
Bases
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Synthesis from Base + ribose to dNDP
dNTP synthesis
dGTP
Note: there is an OH on Caron 3 and a triphosphate on Carbon5 This is
very important!!
Deoxynucleotide triphosphates (dNTP)s are used to DNA.
Of course nucleotide triphosphates are used to synthesize RNA (have
oxygen on C2 of the ribose)
See slide
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Synthesis of DNA (biochemistry)
•Biochemistry of ssDNA synthesis:
•Note that the chain has a phosphate on its 5’ end, and an OH on its 3’
end.
•Addition of dNTP is to 3’ end (ALL nucleic acids are ALWAYS
synthesized by addition to the 3’ end. Therefore synthesis is in the 5’3’
direction.
•Could call the above sequence this 5’-P-GAT-OH-3’, or more simply 5’GAT-3’. Note that 5’-GAT-3’ is different from 5’-TAG-3’.
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Base Pairing
•DNA is usually double stranded and the two strands bind each other via
hydrogen bonding between bases using the BasePairing rules:
A=T two H-bonds (weaker)
G=C Three H-bonds (stronger)
Two Slides
• 5’-GATCT-3’ basepairs with CTAGA:
5’-GATCT-3’
? CTAGA ? but, how is the second strand oriented?
Like this: 5’-GATCT-3’
3’-CTAGA-5’
So, the strands are complimentary and antiparallel !!
If you remember that nucleic acids are synthesized 5’ to 3’ and that they
bind to each other in a complimentary and antiparallel fashion, you should
be able to work out many problems in molecular biol. and this will save
you confusion and lots of memorization!!
DNA structure slide, CAP DNA, lac DNA
Chromosome replication
Initiation of DNA replication:
•Basic idea:
1. Strands separate at a unique site.
2. Replication machinery loads following separation
3. Two new strands are made using old strands as
templates
•Most bacteria have circular chromosomes and replication starts at a site
called “the origin of chromosomal replication” or oriC
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The start of replication is controlled by the protein DnaA
•It recognizes oriC and melts it so that replication enzyme enzymes can
load.
•It also acts as a timer to control when replication starts.
•Therefore it controls WHEN and WHERE replication starts.
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Slides: DnaA1 ,2 ,3,
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Okazaki fragments are ~2000 nt in length and are started by RNA primers
laid down at 5’-GTC-3’ sites.
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Forks1, 2, loop model, Beta, 3D, beta inhibitors, important protiens.
•Resolution of replicated chromosomes by topoisomerase II.
Slide
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Fidelity of DNA replication
•During replication of DNA in E. coli, a permanent mistake is made once
in every 10 billion base pairs (error rate is said to be 10-10 per bp).
The error rate of PolIII during nucleotide addition is about 10-5 per
bp. Therefore there are clearly ways to fix the mistakes made during
synthesis--these result in a ~105 fold decrease in the error rate during
replication.
Example of error rate calculation:
•Assume error rate of 10-10 per bp
Genome size ~7.6 million bp
Average gene size: 1000 bp
1mistake 1000bp 1mistake
x
 7
1010 bp
gene
10 genes
1mistake 7600genes .0008mistakes 1mistake
x


10 7 genes
cell
cell
1300cells

Contributions to decreased error rates:
•The DNA polymerase reaction itself has a an error rate of ~1 mistake per
1000 bp (10-3)
•Proof-reading (3’  5’ exonuclease nuclease activities)reduces that :
•Done by PolIII subunit DnaQ
•A PolI domain dedicated to proofreading
•These contribute ~ 100 fold decrease in error rates
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•A process called methyl-directed mismatch repair contributes to a ~1000
fold further decrease in error rates.
•Total of proof-reading + MDMR is ~105 fold. Enough to bring PolIII
error rate (10-5) to the overall of ~10-10. Other systems are in place to deal
with various types of DNA damage (SOS repair, excision repair,
photoreactivation, etc.)
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Slides of Mut proteins
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Chromosome (genome) organization
Circular (size graph slide)
Linear
Large plasmidsare they plasmids?
GenBank visit to look at chromosome/genome size and organization.
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Chromosome partitioning
Geometry of chromosome partitioning
•Two general models
(Li_MolMicro_2002).
of
chromosome
partitioning
in
E.
coli
Sister chromosome cohesion model: Newly synthesized chromosomes
stay together and are separated late in the cell division cycle:
Extrusion capture model: Chromosomes are partitioned into separate
sides of the cell as they are synthesized (Data supports this one)
Two models for chromosome segregation. A. The extrusion–capture model: after initiation from the central ‘factory site’
(open triangle) the origins (circles) move out toward the poles followed by the newly replicated sequences (thin lines).
Unreplicated DNA (thick line) is fed into the factory, and the terminus (square) is drawn to the cell centre toward the
replication forks (closed triangles). Chromosome markers are segregated progressively as they are replicated, finishing
with the terminus.
Slides from Li et al. (2002) Mol. Micro. 46:985
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Partitioning machinery
•This is still a wide open area of research, but it is becoming clear that there
are several ways to do this. Most of the molecular work has been done
using plasmids (they are partition into daughter cells too) and we will look
at that closely when we discuss plasmids later.
•For now look at the system that partitions the plasmids called P1 and F.
This are single copy plasmids and chromosomes use similar systems for
partitioning chromosomes.
Antibiotics that affect DNA synthesis
Know:
Trimethoprim
Dideoxynucleotides
Ethidium
Nalidixic acid