Download Semiconservative

Bacterial Genetics
The sum total of genetic material of a cell is referred to as the
genome.
The general location and forms of the genome
Chromosome
Subdivided into basic informational packets
called genes
• Procaryotic
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Histonelike proteins condense DNA
Located in cytoplasm
Usually circular
Usually single chromosome
Chromosomes
• Eucaryotic
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–
–
–
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Histone proteins condense DNA
Located in nucleus
Vary in number
Can be diploid (sister chromatids) or haploid
Appear elongate
Genes
• Part of DNA that encodes a protein
• Direct cell function
• Basic unit of heredity
In general, linear sequence of nucleotides or
codons with a fixed start and end point
Genes--> chromosomes-->organisms
Genes
encoded by DNA
• Three categories
– Structural- encode proteins
– Regulatory- control gene expression
– Encode for RNA- rRNA or tRNA
• Genotype
– sum of all gene types
• Phenotype
– Collection or expression of traits encoded in an
organism’s genotype
How genes effect heredity
• Genotype
– sum of all gene types
• Phenotype
– Collection or expression of traits encoded
in an organism’s genotype
Central Dogma
replication
DNA
transcription
RNA
Reverse
transcription
translation
Protein
DNA Structure
• Nucleotide
– Phosphate
– Deoxyribose sugar
– Nitrogenous base
• Double stranded helix
– Antiparallel arrangement
Nitrogenous bases
• Purines
– Adenine
– Guanine
• Pyrimidines
– Thymine
– Cytosine
Purines and pyrimidines pair (A-T or G-C) and the sugars
(backbone) are linked by a phosphate.
Three views of DNA structure
Replication
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Semiconservative
Enzymes
Leading strand
Lagging strand
– Okazaki fragments
Semiconservative
• New strands are synthesized in 5’ to 3’
direction
Semiconservative replication of DNA synthesizes a new strand of
DNA from a template strand.
Simplified steps to show the semiconservative
replication of DNA
Steps in replication
• Uncoiling- unwinding of DNA from histones
• Unzipping- breaking hydrogen bonds
between base pairs allowing strands to
separate
• Addition of nucleotides- each parent strand is
used as the template for synthesis of
daughter strands. Read 3’-5’, synthesis 5’3’
Enzymes
Leading strand
• RNA primer initiates the 5’ to 3’
synthesis of DNA in continuous manner
Lagging strand
• Multiple Okazaki fragments are
synthesized
• Okazaki fragments are ligated together
to form one continuous strand
• Replication begins at the origin of
replication
The steps associated with the DNA replication process.
The bacterial
replicon: a model
for DNA
Synthesis
Replication processes from other biological systems (plasmids,
viruses) involve a rolling cycle.
Simplified model of rolling circle DNA
Replication
RNA
• Transcription
– Message RNA (mRNA)- protein
– Transfer RNA (tRNA)
– Ribosomal RNA (rRNA)
Transcription
• A single strand of RNA is transcribed from a
template strand of DNA
• Template strand- transcribed
Coding strand- nontranscribed
• RNA polymerase catalyzes the reaction
• In eucaryotes
– RNAP I- rRNA
– RNAP II- mRNA -->protein
– RNAP III-tRNA
Eucaryotic mRNA
• mRNA can have interruptions
Gene1
exon
intron
gene 1
exon
•Exon expressed sequences
•Intron intervening sequence
•Removed by splicesosomes. Splicesosome
loops the intron into lariat shape ,excises
them, and joins exons. Some introns
become endonucleases.
The processing of pre-mRNA into mRNA involves the removal of
introns.
RNA
• Thymidine is replaced by uracil
• Synthesis in 5’ to 3’ direction (with
regard to RNA)
• The message contains a codon (three
bases)
Transcription
• Initiates when RNAP recognizes a
promoter region
Two regions
– 1) 35 bp upstream from transcription start
site
– 2) 10 bp upstream from transcription start
site
Transcription
• Terminates when RNAP reaches
terminators in sequence. These make
RNAP stutter and fall off or pause and
fall off
The synthesis of mRNA from DNA.
The major events in mRNA synthesis
Transcription regulation
• Operons- coordinated set of genes
which are regulated as a single unit.
They are transcribed as a single unit.
• Types of operons
– Inducible
– Repressible
• Regulator gene- gene that can repress
the operon
• Operator- acts as on/off switch
• Structural gene- genes to be transcribed
when the operon is on
Lactose operon (inducible)
• Breaks down lactose
• Normally in OFF position. Operon is
controlled by the binding of the repressor to
the operator
• Lactose binding to repressor causes
conformation changes in the repressor
• Repressor dislodges from the operator
• RNAP binds and transcribes structural genes
• When lactose levels fall: the repressor is free
to bind the operator again
• In this system lactose is the inducer
• This operon is not inducible in the presence
of glucose. Glucose is preferred over lactose
as a carbon source
The regulation of sugar metabolism such as lactose involves
repression in the absence of lactose, and induction when lactose
is present.
The lactose operon in bacteria
Repressible operon
• Operon is normally on
• Corepressor- normally the product of
the operon. Turns operon off by binding
and activating the repressor
The regulation of amino acids such as arginine involves
repression when arginine accumulates, and no repression when
arginine is being used.
Repressible operon
mRNA
• Copy of a structural gene or genes of
DNA
– Can encode for multiple proteins on one
message
• Thymine is replaced by uracil
• The message contains codons
• Carries code for proteins
tRNA
• Copy of specific regions of DNA
• Cloverleaf structure
• Complimentary sequences form hairpin loops
– Amino acid attachment site
– Anticodon
• Participates in translation (protein synthesis)
– Delivers amino acid to growing polypeptide chain
Important structural characteristics for tRNA and mRNA.
Characteristics of transfer and message RNA
rRNA
• Consist of two subunits (70S)
• A subunit is composed of rRNA and
protein
• Participates in translation
Ribosomes bind to the mRNA, enabling tRNAs to bind, followed
by protein synthesis.
Summary of the flow
of genetics
Codons
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•
•
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Triplet code that specifies a given amino acid
Multiple codes for one amino acid
20 amino acids
Start codon
Stop codons
Redundant code allows for wobble
The codons from mRNA specify a given amino acid.
The Genetic code
Representation of the codons and their corresponding amino
acids.
Protein
• Translation
– Protein synthesis have the following
participants
• mRNA
• tRNA with attached amino acid
• Ribosome
Participants involved in the translation process.
The “players” in translation
Translation
• Ribosomes bind mRNA near the start codon
(ex. AUG)
• tRNA anticodon with attached amino acid
binds to the start codon
• Ribosomes move to the next codon, allowing
a new tRNA to bind and add another amino
acid
• Series of amino acids form peptide bonds
• Stop codon terminates translation
The process of translation.
The events in protein synthesis
For procaryotes, translation can occur at multiple sites on the
mRNA while the message is still being transcribed.
Speeding up the protein assembly line in bacteria
Transcription and translation
in eucaryotes
• Similar to procaryotes except
– AUG encodes for a different form of
methionine
– mRNA code for one protein
– Transcription and translation are not
simultaneous
– Pre-mRNA
• Introns
• Exons
Mutations
• Loss of bases
• Addition of bases
• Misincorporation of bases
• Wild-type: natural, nonmutated strain
• Mutant: harbors 1 or more mutations
Mutations
• Changes made to the DNA
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Spontaneous – random change
Induced – chemical, radiation.
Point – change a single base
Nonsense – change a normal codon into a stop
codon
– Back-mutation – mutation is reversed
– Frameshift – reading frame of the mRNA changes
Examples of chemical and radioactive mutagens, and their
effects.
Selected mutagenic agents and their effects
Ames test
Salmonella culture
(his-) cannot make histidine
Enriched media (his+)
18 colonies
Minimal media (his-)
3 colonies
TOTAL BACTERIA
SPONTANEOUS
MUTANTS
Minimal media (his-)
+ test reagent
6 colonies
INDUCED + SPONTANEOUS
MUTANTS
Effects of mutations
• Positive effects for the cell
– Allow cells to adapt
• Negative effects for the cell
– Loss of function
– Cells cannot survive
Recombination
• Sharing or recombining parts of their
genome
– Conjugation
– Transformation
– Transduction
Conjugation
• Transfer of plasmid DNA from a F+ (F factor)
cell to a F- cell
• An F+ bacterium possesses a pilus
• Pilus attaches to the recipient cell and creates
pore for the transfer DNA
• High frequency recombination (Hfr) donors
contain the F factor in the chromosome
Conjugation is the genetic transmission through direct contact
between cells.
Conjugation: genetic transmission through direct contact
Transformation
• Nonspecific acceptance of free DNA by
the cell (ex. DNA fragments, plasmids)
• DNA can be inserted into the
chromosome
• Competent cells readily accept DNA
DNA released from a killed cell can be accepted by a live
competent cell, expressing a new phenotype.
Griffith’s classic experiment in transformation
Transduction
• Bacteriophage infect host cells
• Serve as the carrier of DNA from a
donor cell to a recipient cell
– Generalized
– Specialized
Genetic transfer based on generalized transduction.
Generalized transduction
Genetic transfer based on specialized transduction.
Specialized transduction
Transposon
• “Jumping genes”
• Exist in plasmids and chromosomes
• Contains genes that encode for
enzymes that remove and reintegrate
the transposon
• Small transposons are called insertion
elements