Transcript Genetic mechanisms

Microbial Genetics
Chapter 9
transmission of biological traits from
parent to offspring
2. expression & variation of those traits
3. structure & function of genetic material
4. how this material changes
1.
Genetics – the study of heredity
Levels of genetic study
What you need to know about
microbial genetics…
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Genetic structure/function – of DNA,
chromosomes, genes and genomes; also
including size and arrangement both
prokaryotes and eukaryotes.
Mechanisms of replication, transcription
and translation including enzymes for
proks., euks. and viruses.
Gene regulation: inducible vs. repressible
operons (Prokaryotes only)
Mutations – types, causes and effects
Recombination – conjugation,
transformation.
Famous names in the history of genetics
Genetic structure/function
Levels of structure & function of the genome
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____________ – sum total of genetic material
of an organism (chromosomes +
mitochondria/chloroplasts and/or plasmids)
◦ genome of cells – DNA
◦ genome of viruses – DNA or RNA
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____________– length of DNA containing
genes
____________ -fundamental unit of
heredity responsible for a given trait
◦ site on the chromosome that provides
information for a certain cell function
(____________ )
◦ segment of DNA that contains the necessary code
to make a protein or RNA molecule
Genetic structure/function
Locations and forms of genomes
Genomes vary in size
Genetic structure/function
smallest virus – 4-5 genes
 E. coli – single chromosome containing
4,288 genes; 1 mm; 1,000X longer than
cell (~4.5 Mbp)
 Human cell – 46 chromosomes containing
31,000 genes; 6 feet; 180,000X longer
than cell
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Genetic structure/function
Prokaryotes: coiled into tight bundle by
gyrase (a topoisomerase)
 Eukaryotes:
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1. wound around histone proteins to form
nucleosomes
2. nucleosomes condense, coil into chromatin
fibers
3. Chromatin supercoils and condenses into
__________________
Genome packaging
Genetic structure/function
Genetic structure/function
Nucleic acid structure (RNA, DNA)
Nucleic acids are made of nucleotides
(polymer, monomer)
 each nucleotide consists of 3 parts
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◦ a 5 carbon _____________(deoxyribose or
ribose)
◦ a __________________ group
◦ a nitrogenous base (adenine, thymine,
cytosine, guanine, and uracil)
◦ Purines: A + G; Pyrimidines: C, U, T
Genetic structure/function
DNA structure
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2 strands twisted into a double helix
sugar -phosphate backbone
nitrogenous bases form steps in ladder (inside
helix)
◦ constancy of base pairing (purines pair with
pyrimidines)
◦ _______________ with 2 hydrogen bonds
(RNA – A with U)
◦ __________________ with 3 hydrogen bonds
antiparallel strands __________________
each strand provides a template for the exact
copying of a new strand
 order of bases constitutes the DNA code
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Genetic structure/function
Genetic structure/function
Genetic structure/function
Torsion in helix and “steps” of nucleotides
results in major and minor grooves in helix
Genetic structure/function
Significance of DNA structure
Maintenance of code during reproduction.
Constancy of base pairing guarantees
that the code will be retained.
2. Providing variety. Order of bases
responsible for unique qualities of each
organism (gene sequence)
1.
Possible arrangement of nucleotides is nearly
infinite: 4n where n=#nucleotides. So for a
1000 bp gene, 4n combinations = 41000 or 1.5
x 10602 !!
Genetics - History
__________________________ – 1944
– showed DNA was the molecule carrying the
blueprint for life. Won Nobel prize.
 Erwin Chargaff – components of DNA
 Maurice Wilkins and
______________________– XRay
crystallography gave clue to double helix
structure
 Structural model: credit (Nobel prize) goes
to
History
of DNA structure
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_- 1953
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DNA replication
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Genetic mechanisms
-Replication
Due to base pairing, each strand serves as
template for the synthesis of a new strand
DNA replication is
________________because each
chromosome ends up with one new strand of
DNA and one old strand.
DNA – A with T, G with C – so one strand can be template for its complement strand
Semi-conservative replication of
DNA
Genetic mechanisms
-Replication
Genetic mechanisms
-Replication
DNA replication (bacteria)
Steps:
 Begins at an origin of replication
 ___________unwinds and unzips the
DNA double helix (replication fork)
 Strands are kept separate by SSBP (single
strand binding proteins)
 An RNA ___________is synthesized
(primase) and “primes” (DNA polymerase
III cannot initiate synthesis on its own)
Replication (cont’d)
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Genetic mechanisms
-Replication
DNA polymerase III adds nucleotides in a 5’
to 3’ direction – works on both strands
at once (see movie!)
◦ Leading strand – synthesized continuously in 5’
to 3’ direction
◦ Lagging strand – synthesized 5’ to 3’ in short
segments (Okazaki fragments);
 overall direction of DNA pol III movement is 3’ to 5’
___________– removes primers (RNA)
and replaces with DNA
 ___________– fills in gaps/nicks
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Genetic mechanisms
-Replication
Enzymes involved in DNA
replication (short list)
Bacterial replicon
Genetic mechanisms
-Replication
(origin is A/T rich, easy to
separate)
SEE THE TWO MOVIES ON THE WEB SITE!!
Genetic mechanisms
-Replication
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Separation of
daughter
molecules occurs
via a “nick” which
is then repaired
Two completed
molecules will go
to daughter cells
(binary fission)
Genetic mechanisms
-Replication
Eukaryotes – similar to this but there are
thousands of replicons acting
simultaneously (replication bubbles)
 Rolling circle replication – small circular
genetic material (plasmids)
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Other types of replication
genetic
information
Flow of genetic information
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What are the products that genes encode?
◦ Structural genes – code for proteins
◦ Genes that code for RNA (they are not translated!)
◦ Regulatory genes that control the expression of
other genes
___________– is an organisms genetic
makeup
 ___________– is the physical trait that
results from the expression of the organism’s
genes
 How are genes expressed?
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◦ transcription and translation
Gene expression
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Genetic mechanisms
-Gene expression
________________– DNA template is used
to synthesize RNA (transcript)
◦ ______________________is the enzyme
responsible
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________________–making a protein
using the information provided by messenger
RNA (mRNA) – involves decoding the mRNA
◦ occurs on ribosomes, and involves tRNA and amino
acids
◦ Proteins – end product of gene expression
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Please view transcription + translation
movies on the web page
Genetic mechanisms
-Gene expression
Each triplet of nucleotides (codon)
specifies a particular amino acid.
• structure  function A protein’s
primary structure determines its shape
& function.
• Proteins determine phenotype. Living
things are what their proteins make
them.
• DNA is mainly a blueprint that tells the
DNA-protein
relationship
cell which kinds
of proteins to make
and how to make them.
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Genetic mechanisms
-Gene expression
DNA-protein relationship
Genetic mechanisms
-Gene expression
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Three types:
messenger RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
How does RNA differ from DNA?
Uses Uracil (U) instead of Thymine (T)
Single stranded (except in some viruses)
Ribose is the sugar
RNA
DNA
Genetic mechanisms
-Gene expression
Transcription
RNA polymerase
RNA
Translation
ribosomes
PROTEINS
Genetic mechanisms
-Gene expression
RNA polymerase binds to promoter region
upstream of the gene
2. RNA polymerase adds nucleotides
complementary to the template strand of a
segment of DNA in the 5’ to 3’ direction
(downstream of promoter)
3. Uracil is placed as adenine’s complement (U
with A)
4. At termination, RNA polymerase recognizes
signals and releases the transcript
 Transcription
100-1,200 bases long
- steps
1.
Transcription
Genetic mechanisms
-Gene expression
DNA
Genetic mechanisms
-Gene expression
Transcription
RNA polymerase
RNA
Translation
ribosomes
PROTEINS
Genetic mechanisms
-Gene expression
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Ribosomes assemble on the ___________of
a mRNA transcript
Ribosome scans the mRNA until it reaches the
start codon, usually _______(met)
A tRNA molecule with the complementary
anticodon and methionine amino acid enters
the P site of the ribosome & binds to the
mRNA
mRNA triplet code is translated into amino
acids (elongation)
Translation - intiation
Translation
Genetic mechanisms
-Gene expression
Genetic mechanisms
-Gene expression
Genetic mechanisms
-Gene expression
Genetic mechanisms
-Gene expression
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A second tRNA with the complementary
anticodon fills the A site
A peptide bond is formed
The first tRNA is released and the ribosome
slides down to the next codon (5’3’ reading
frame = triplet).
Another tRNA fills the A site & a peptide bond
is formed.
This process continues until a stop codon is
encountered.
Translation elongation
Genetic mechanisms
-Gene expression
Termination (________) codons – UAA,
UAG, and UGA – are codons for which
there is no corresponding tRNA.
 When this codon is reached, the ribosome
falls off and the last tRNA is removed
from the polypeptide.
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Translation termination
Polyribosomal complex =
transcription and multiple translation
simultaneously (bacteria)
Genetic mechanisms
-Gene expression
Eucaryotic
transcription
&Transcription
1. Do not occur
simultaneously.
translation
from
procaryotic
occurs in differs
the nucleus
and translation
occurs
2.
3.
4.
in the cytoplasm.
Eucaryotic start codon is AUG, but it does
not use formyl-methionine.
Eucaryotic mRNA encodes a single protein,
unlike bacterial mRNA which encodes many
(operon).
Eucaryotic DNA contains introns –
intervening sequences of noncoding DNAwhich have to be spliced out of the final
mRNA transcript.
Split gene of eucaryotes
Genetic mechanisms
-Gene expression
Genetic mechanisms
-Gene expression
Some transcribed genes
aren’t translated
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Genes encoding for RNAs such as tRNA,
rRNA, RNA primers (used in DNA
replication)
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These RNAs have 2ndary structure like
proteins but function as RNA
Regulation of protein
synthesis & metabolism
Operons
Gene regulation
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a coordinated set of genes, all of which are
regulated as a single unit. Found in
prokaryotes.
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2 types – based on regulation
◦ ________________– operon is turned ON by
substrate: catabolic operons- enzymes needed to
metabolize a nutrient are produced when needed
◦ ________________– operon is turned OFF by the
product synthesized; anabolic operon –enzymes
used to synthesize an amino acid stop being
produced when enough is made
Lactose operon: inducible
operon
Gene regulation
Made of 3 segments:
1. ________________gene that codes for
________________
2. ___________locus- composed of
promoter and operator
3. ___________locus- made of 3 genes
each coding for an enzyme needed to
catabolize lactose –
b-galactosidase – hydolyzes lactose (gal and glu)
permease - brings lactose across cell membrane
b-galactoside transacetylase – uncertain function
Gene regulation
Lac operon: inducible
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________________
◦ In the absence of lactose the repressor binds
with the operator locus and blocks transcription
of downstream structural genes
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Lactose
____________________________
◦ Binding of lactose to the repressor protein
changes its shape and causes it to fall off the
operator. RNA polymerase can bind to the
promoter. Structural genes are transcribed.
Lactose operon
1. Repressor (protein) is a product
of a regulator gene elsewhere in
the genome
2. It is attached to operator in
absence of substrate and blocks
transcription of genes
“downstream”
1. Lactose (substrate, inducer) binds
repressor
2. Repressor changes shape, comes
off operator
3. RNA pol can now transcribe gene
4. Enzymes for lactose catabolism
are translated
5. When lactose levels go back
down, repressor will bind to
operator again
Gene regulation
Gene regulation
Normally on and will be turned off when
product is no longer needed (excess).
 When excess arginine is present, it binds
to the repressor and changes it. Then the
repressor binds to the operator and blocks
arginine synthesis.
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Arginine operon: repressible
Repressible operon
1. Repressor isn’t attached to
operator, so operon transcription
is continuous (genes are “on”)
and ARG is being made
2. This continues is as long as ARG
is being used by the cell
1. Excess product (ARG) builds up
from not being used by cell
2. ARG (corepressor) binds
repressor which changes shape,
and binds operator
3. RNA pol cannot transcribe gene
4. When ARG levels get too low,
operator will fall off and
transcription will begin again
Antibiotics that affect gene expression
Rifamycin – binds to RNA polymerase
Actinomycin D - binds to DNA & halts mRNA
chain elongation
 Erythromycins – interfere with attachment
of mRNA to ribosomes
 Chloramphenicol, linomycin &
tetracycline - bind to ribosome and block
elongation
 Streptomycin – inhibits peptide initiation &
elongation
 Problem with drugs that affect prokaryotic
ribosomes is that they affect
________________too
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Mutations – change in
______________ sequence
 ________________– addition of genes
from an outside source (another cell,
another organism)
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Changing the genetic code
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Any permanent, inheritable change in genetic
information
Alteration of the nucleotide sequence (ATGC)
Involves either loss, addition or
rearrangement of base pairs
Spontaneous mutation – random, due to
replication error
Induced mutation – result from exposure to
mutagens (physical, chemical; disrupt DNA)
Mutations
mutations
Types of Mutations
________________mutation – addition,
deletion or substitution of a few bases
 ________________mutation – causes
change in a single amino acid
 ________________mutation – changes
a normal codon into a stop codon
 ________________mutation – alters a
base but does not change the amino acid
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Excisio
n repair
Mutations
-repair
Ames Test
mutations
Also
lacks
DNA
repair
enzym
es and
has
leaky
cell
walls
Chemicals that
produce an increased
# of back mutations
(more than
spontaneous) are
considered mutagens
Spontaneous
Back-mutation
Induced
Back-mutation
Genetic Recombination
Types of intermicrobial exchange
Recombination events: genetic transfer resulting in a new strain different from
both parent strains
requires the attachment of two
related species & formation of a
bridge that can transport DNA
transfer of naked DNA
transduction
DNA transfer mediated by bacterial
virus
Conjugation
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_____________– transfer of a
plasmid or chromosomal fragment from
a donor cell to a recipient cell via a
direct connection
◦ Gram-negative cell donor has a fertility
plasmid (F plasmid, F′ factor) that allows
the synthesis of a conjugative pilus
◦ Recipient cell is a related species or genus
without a fertility plasmid
◦ Donor transfers fertility plasmid to recipient
through pilus
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Genetic Recombination
-Conjugation
Figure 9.23 (2)
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_____________
High-frequency recombination – donor’s
fertility plasmid has been integrated
into the bacterial chromosome
 When conjugation occurs, a portion of
the chromosome and a portion of the
fertility plasmid are transferred to the
recipient
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Figure 9.23 (3)
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Transformation
Genetic Recombination
-transformation
_____________– chromosome
fragments from a lysed cell are accepted
by a recipient cell; the genetic code of
the DNA fragment is acquired by the
recipient
 Donor and recipient cells can be
unrelated
 Useful tool in recombinant DNA
technology
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Genetic Recombination
-transformation
Figure 9.24
Insert figure 9.23
transformation
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Consequences of changing the genetic code
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Anything that alters the DNA sequence
(Mutations, transposons, transformation,
etc.) can be beneficial or harmful to the
microbe
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Examples:
◦ Recombination events can result in increased fitness
to the microbe, such as gaining antibiotic resistance,
virulence factors etc.
◦ Some mutations or disruptions of genes (transposons)
can be lethal to the cell if they interrupt crucial
genes (like those involved with metabolism)
◦ Remember - Mutations – cause harm if they change
the final protein product to a non-functional protein.
So if the AA is the same, the protein is not changed.
(example: AUG  AUA ; Both are methionine) (Silent
mutations)