Gene Structure, Replication & Expression Flashcards
DNA replication
*Unwind the DNA helix (helicase) and use each strand as a template for DNA polymerization (DNA polymerase III, replication of chromosome)
*Replication fork (replisome / 30 proteins)
*Semiconservative: -one parent strand -one replica
*DNA polymerases always synthesize DNA in the 5’ to 3’ direction using dNTPs
*DNA polymerases have a 3’ to 5’ exonuclease activity (proofreading)
Replication of Bacterial DNA(chromosomes and plasmids)
only one origin (A-T rich region, easier to break bond)
2 replisome
1 terminus(usually opposite side of Origin)
Rollong -Circle Replication
some conjugative plasmids
-origin recognize by an enzyme, make a nick on one of the two strands
-3’OHrecognize by DNA polymerase, start synthesis, peel off the strand
-DNA Pol go multiple times around the plasmid, generates very long linear pieces of DNA
-Second strands synthesize by another DNA pol, cut into one unit, religate together
Replication of E DNA
multiple origin and initiation
Mechanism of DNA Replication in P
DnaA helicase binds to oriC locus (AT rich region)(need ATP)
after seperatre, DnaB replaces DnaA (replication fork: 75-100 rev/sec)
the whole replisome construct around Dna B. in the replication fork, Dna B is the helicase
Topoisomreases(DNA gyrase): release the tension created by the helicases unwinding DNA, cut phosphorester bond, religate
Single-stranded DNA binding proteins (SSBs)keeps two strands seperated
DNA polymerase III replicates the two strands in the 5’ to 3’ direction (750-1000 base pairs/second)
DNA pol 1
degrade RNA priers and synthesize DNA to replace the RNA primer
Leading Strand Synthesis
DNA polymerase III synthesizes the second DNA strand 5’ to 3’ continuously
At the beginning, their is a primer get synthesized, DNA pol recognize the primer and start synthesize
Lagging Strand Synthesis
*Primase (RNA polymerase) synthesizes a short RNA (10 nt) complementary to DNA
*DNA polymerase III uses the RNA as a primer and synthesizes the second DNA strand 5’ to 3’ (discontinuous)
*Okazaki fragments (1000-2000 nt in bacteria) (100 nt in eucaryotes)
*DNA poly 1: The RNA primer is degraded (3’-5’ exonuclease), the gap is filled, and the fragments are joined (DNA ligase)
DNA pol 3 hit the primer and replaced by DNA pol1
What is a Gene (also called?)
cistron
A nucleotide sequence that codes for an mRNA (protein), tRNA, or rRNA.
*Eucaryotic genes are mostly interrupted by introns (noncoding sequence)
*Bacterial genes are mostly continuous (some have intron)
*Each amino acid is specified by three consecutive nucleotides called a codon.
Most bacterial genes have at least four parts, each with different functions
promoter
leader
coding region
and trailer
Splicing
splicing intron
occur in mRNA
Codon
A codon cannot be shorter then 3 nucleotides
*The code is degenerate; more than one codon for most amino acids (up to 6)
*Two amino acids have only one codon-Tryptophan (Trp), methionine (Met)
*There is 61 sense codons and 3 stopcodons (nonsense codons)
*There are not 61 different tRNAs because of the wooble (loose pairing) pairing between the codon (mRNA) and anticodon (tRNA) during translation
Wooble and Coding
Glycine mRNA codon: GGU, GGC, GGA, GGG
tRNA anticodon: ICC, CCC
isosine: C=O replace the amino group on adenine, can bind with A C U
Organization of Bacterial Gene
-35
-10
+1
Sequence in leader region
-35: RNA pol recog site
TTGACA
-10: RNA pol binding site
TATA box or Pribnow box
TATAAT
+1: transcription start AUG
shine-Dalgarno sequence: ribosome recog & binding site
Mutation type in DNA (5)
base substitution
deletion
inversion
transposition
duplication
what kind of change will mutation cause
*Morphological mutations
–colonial or cellular morphology
*Lethalmutations (essential genes)
*Biochemicalmutations (biosyntheticpathways)
–prototrophto & auxotroph
*Resistantmutant
–Antibiotics, chemicals
reasons for mutation (7)
*Spontaneous (DNA Replication)
error didn’t fixed
*Induced by mutagens
–physical (e.g., U.V., X rays, grays)
–chemical (e.g., 5-bromouracil)
*Point mutations (only 1 base pair):
–silent (do not change codon)
–missense (change codon)
–nonsense (create a stop codon)
–frameshift (deletions and insertions
missense, nonsense, and frameshift will cause amino acids change
Mutations direction in DNA
*Forward mutation (from the wild-type)
*Reversion mutation (back to wild-type phenotype, gene sequence might be different)
–Original wild-type sequence
–New codon but same amino acid(silent)
–New codon but similar amino acid(missense)
*Suppressor mutation
–Intragenic(same gene but at another location)
-Extragenic (different gene)
Intragenic and example
same gene but at another location
Suppressor mutation
mutation in RNA pol enzyme
cause pol to be inactive, another mutation some where else in the gene, change another amino acids active site, reactive the enzyme
Extragenic and example
different gene
Suppressor mutation
mutation let DNA pol be inactive, another mutation in the protein binds with DNA pol have a mutation, reactivate DNA pol
DNA splippage
Slippage cause mutation
导致多or少bp
DNA pol mistake？
Tymine dimer
caused by UV
covalent link
if too much, will cause cancer
5-bromouracil
50% keto form: bind with Adenine (c=o)
50% enol form: bind with guanine (c-o)
form is random
DNA repair
*Proofreading activity of DNA polymerases(3’ to 5’ exonuclease activity)
fix point mutation, most of time, mutation will be removed and the chain will resynthesize
*Mismatch Repair
–not complementary base pairs (e.g. G=T)
check follow replication
*Nucleotide excision repair
–distorted DNA (e.g. thymine dimer)
*Base excision repair
–apurinic(A, G) and apyrimidinic(C, T) sites
–damaged or unnatural bases (have U in DNA)
Mismatch repiar
Mut S:
Bind with DNA follow replication, recognize the incorrect base pairs
when recognizing base pairs, recruit L & H
Mut L and Mut H:
all are endonuclease
Mut H cut the newly synthesize strand
the strand removed by exonuclease
DNA poly III synthesis a new strand
How to know cut with strand in mismatch repair?
Bacteria DNA is methylated
the newly synthesized strand is not methylated yet
Nucleotide excision repair
Uvr A and Uvr B bind to DNA and scan the DNA distorted structure
recognized the thymine dimer
Uvr A replaced by Uvr C
UvrC: endonuclease, cut on both side of the damage region
Uvr D: helicase, remove the damage region
DNA pol I: recognize the 3’end and resynthesize the DNA
DNA ligase seal the gap
Base excision repair
DNA glycosylase: recognize the abnormal gene, cleave the bond between base and sugar
AP endonuclease: recognize a missing base and cleave the DNA backbone on the 5’ side of the missing base
DNA pol I use it 5’-3’exonuclease activity to remove the damage and resynthesize
DNA ligase seal
3’ to 5’ exonuclease activity
5’ to 3’ exonuclease activity
DNA pol I
The 3’ to 5’ can only remove one mononucleotide at a time, and the 5’ to 3’ activity can remove mononucleotides or up to 10 nucleotides at a time
DNA pol III
3’–5’ activity
reading 3’→5’ and synthesizing 5’→3’.
DNA repair 2 different types of system
Direct removal of lesions (error free systems)
does not involve in DNA synthesis
–thymine dimers (photoreactivation, photolyase)
–methyls(methylguanine methyltransferase)
–alkyls (alkyltransferase)
*Recombination Repair (recAprotein)
–deal with complex mutations at same position
*double strand breaks
*multiple lesions
recombinational repair
by swap stands, recombine
separate mutation to different strand
can be fix by other mechanism
just before division, 2 genome, allow recombination occur
