Microbial Genetics
2
Genetics and Genes
Genetics – the study of heredity
The science of genetics explores:
1. Transmission of biological traits from parent
to offspring
2. Expression and variation of those traits
3. Structure and function of genetic material
4. How this material changes
3
Levels of genetic study
4
Levels of Structure and Function of
the Genome
• Genome – sum total of genetic material of an
organism (chromosomes + mitochondria/chloroplasts
and/or plasmids)
– genome of cells – DNA
– genome of viruses – DNA or RNA
• DNA complexed with protein constitutes the genetic
material as chromosomes.
• Bacterial chromosomes are a single circular loop.
• Eukaryotic chromosomes are multiple and linear.
5
Chromosome is subdivided into genes, thefundamental 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
Three basic categories of genes:
1. Genes that code for proteins - structural genes
2. Genes that code for RNA
3. Genes that control gene expression - regulatory genes
6
• All types of genes constitute the genetic
makeup – genotype.
• The expression of the genotype creates
observable traits – phenotype.
7
Genomes Vary in Size
• Smallest virus – 4-5 genes
• E. coli – single chromosome containing
4,288 genes; 1 mm; 1,000X longer than cell
• Human cell – 46 chromosomes containing
31,000 genes; 6 feet; 180,000X longer than
cell
8
9
10
DNA• Two strands twisted into a helix
• Basic unit of DNA structure is a nucleotide
• Each nucleotide consists of 3 parts:
– a 5 carbon sugar - deoxyribose
– a phosphate group
– a nitrogenous base – adenine, guanine, thymine, cytosine
• Nucleotides covalently bond to form a sugar-phosphate linkage – the backbone
– each sugar attaches to two phosphates –
• 5′ carbon and 3′ carbon
11
DNA
• Nitrogenous bases covalently bond to the 1′ carbon of each sugar and span the center of the molecule to pair with an appropriate complementary base on the other strand
– adenine binds to thymine with 2 hydrogen bonds
– guanine binds to cytosine with 3 hydrogen bonds
• Antiparallel strands 3′ to 5′ and 5′ to 3′
• Each strand provides a template for the exact copying of a new strand
• Order of bases constitutes the DNA code
12
13
Significance of DNA Structure
1. 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.
14
DNA Replication
• Making an exact duplicate of the DNA involves 30 different enzymes
• Begins at an origin of replication
• Helicase unwinds and unzips the DNA double helix
• An RNA primer is synthesized by primase
• DNA polymerase III adds nucleotides in a 5′ to 3′ direction
– leading strand – synthesized continuously in 5′ to 3′ direction
– lagging strand – synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′
15
• DNA polymerase I removes the RNA
primers and replaces them with DNA.
• When replication forks meet, ligases link
the DNA fragments along the lagging strand
to complete the synthesis.
• Separation of the daughter molecules is
complete.
16
17
As replication proceeds
one strand loops down
and final separation
occurs when enzyme cuts
and releases two
completed molecules.
DNA replication is semiconservative because
each chromosome ends up with one new
strand of DNA and one old strand.
19
Applications of the DNA code
• Information stored on the DNA molecule is
conveyed to RNA molecules through the
process of transcription.
• The information contained in the RNA
molecule is then used to produce proteins in
the process of translation.
20
21
Gene-Protein Connection
1. Each triplet of nucleotides on the RNA specifies a particular amino acid.
2. A protein’s primary structure determines its shape and function.
3. Proteins determine phenotype. Living things are what their proteins make them.
4. DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them.
22
23
RNAs
• Single-stranded molecule made of nucleotides
– 5 carbon sugar is ribose
– 4 nitrogen bases – adenine, uracil, guanine, cytosine
– phosphate
24
RNA• 3 types of RNA:
– messenger RNA (mRNA) – carries DNA message
through complementary copy; message is in triplets
called codons
– transfer RNA (tRNA) – made from DNA;
secondary structure creates loops; bottom loop
exposes a triplet of nucleotides called anticodon
which designates specificity and complements
mRNA; carries specific amino acids to ribosomes
– ribosomal RNA (rRNA) – component of ribosomes
where protein synthesis occurs
25
26
Transcription
1. 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.
3. Uracil is placed as adenine’s complement.
4. At termination, RNA polymerase recognizes signals
and releases the transcript.
• 100-1,200 bases long
27
28
Translation
• Ribosomes assemble on the 5′ end of a mRNA transcript.
• Ribosome scans the mRNA until it reaches the start codon, usually AUG.
• A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mRNA.
29
30
Translation Elongation
• 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.
• Another tRNA fills the A site and a peptide bond is formed.
• This process continues until a stop codon is encountered.
31
Translation Termination
• 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.
32
33
The Master Genetic Code
• Represented by the mRNA codons and the
amino acids they specify
• Code is universal
• Code is redundant
34
35
36
Polyribosomal complex allows for the synthesis of
many protein molecules simultaneously from the
same mRNA molecule.
37
Eucaryotic Transcription and
Translation
1. Do not occur simultaneously – transcription occurs in the nucleus and translation occurs in the cytoplasm.
2. Eucaryotic start codon is AUG, but it does not use formyl-methionine.
3. Eucaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many.
4. Eucaryotic DNA contains introns – intervening sequences of noncoding DNA- which have to be spliced out of the final mRNA transcript.
38
39
Genetics of Animal Viruses
• Viral genome - one or more pieces of DNA
or RNA; contains only genes needed for
production of new viruses
• Requires access to host cell’s genetics and
metabolic machinery to instruct the host cell
to synthesize new viral particles
40
41
42
Regulation of Protein Synthesis and
Metabolism
• Genes are regulated to be active only when
their products are required.
• In procaryotes this regulation is coordinated
by operons, a set of genes, all of which are
regulated as a single unit.
43
Operons
• 2 types of operons:
– inducible – operon is turned ON by substrate:
catabolic operons- enzymes needed to metabolize
a nutrient are produced when needed
– repressible – genes in a series are turned OFF by
the product synthesized; anabolic operon –
enzymes used to synthesize an amino acid stop
being produced when they are not needed
44
Lactose Operon: Inducible Operon
Made of 3 segments:
1. Regulator- gene that codes for repressor
2. Control locus- composed of promoter and
operator
3. Structural locus- made of 3 genes each coding
for an enzyme needed to catabolize lactose –b-galactosidase – hydolyzes lactose
permease - brings lactose across cell membrane
b-galactosidase transacetylase – uncertain function
45
Lac Operon
• Normally off
– In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes.
• Lactose turns the operon on.
– 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.
46
47
Arginine Operon: Repressible
• Normally on and will be turned off when
nutrient is no longer needed
• When excess arginine is present, it binds to
the repressor and changes it. Then the
repressor binds to the operator and blocks
arginine synthesis.
48
49
Antibiotics That Affect Transcription
and Translation
• Rifamycin – binds to RNA polymerase
• Actinomycin D - binds to DNA and halts mRNA chain elongation
• Erythromycin and spectinomycin – interfere with attachment of mRNA to ribosomes
• Chloramphenicol, linomycin and tetracycline-bind to ribosome and block elongation
• Streptomycin – inhibits peptide initiation and elongation
50
Mutations: Changes in the Genetic Code
• A change in phenotype due to a change in genotype (nitrogen base sequence of DNA) is called a mutation.
• A natural, nonmutated characteristic is known as a wild type (wild strain).
• An organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc.
51
Causes of Mutations
• Spontaneous mutations– random change
in the DNA due to errors in replication that
occur without known cause
• Induced mutations – result from exposure
to known mutagens, physical (primarily
radiation) or chemical agents that interact
with DNA in a disruptive manner
52
Categories of Mutations
• Point mutation – addition, deletion or
substitution of a few bases
• Missense mutation – causes change in a
single amino acid
• Nonsense mutation – changes a normal
codon into a stop codon
• Silent mutation – alters a base but does not
change the amino acid
53
Categories of Mutations
• Back-mutation – when a mutated gene
reverses to its original base composition
• Frameshift mutation – when the reading
frame of the mRNA is altered by the
addition or deletion of nucleotides in a
newly synthesized DNA
54
Repair of Mutations
• Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA.
– DNA polymerase – proofreads nucleotides during DNA replication
– Mismatch repair – locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase
– Light repair – for UV light damage
– Excision repair – locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides
55
56
The Ames Test
• Any compound known to be mutagenic is
considered to be carcinogenic.
• Agricultural, industrial, and medicinal
compounds are screened using the Ames test.
• Indicator organism is a mutant strain of
Salmonella typhimurium that has lost the ability
to synthesize histidine.
• This mutation is highly susceptible to back-
mutation.
57
58
Positive and Negative Effects Of
Mutations
• Mutations leading to nonfunctional proteins are
harmful, possibly fatal.
• Organisms with mutations that are beneficial in
their environment can readily adapt, survive, and
reproduce – these mutations are the basis of
change in populations.
• Any change that confers an advantage during
selection pressure will be retained by the
population.
59
DNA Recombination Events
Genetic recombination – occurs when an
organism acquires and expresses genes
that originated in another organism
3 means for genetic recombination in bacteria:
1. Conjugation
2. Transformation
3. Transduction
60
Conjugation
• Conjugation – 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 conjugation (sex) pilus
– recipient cell is a related species or genus without a fertility plasmid
– donor transfers fertility plasmid to recipient through pilus
61
62
Conjugation
• 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.
63
64
Transformation
• 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
65
Insert figure 9.23transformation
66
Transduction
• Transduction – bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell
• Two types:
– generalized transduction – random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way
– specialized transduction – a highly specific part of the host genome is regularly incorporated into the virus
67
68
69
Transposons
• Special DNA segments that have the capability of moving from one location in the genome to another – “jumping genes”
• Cause rearrangement of the genetic material
• Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome
• May be beneficial or harmful
70