Download Chapter 8 Lecture Notes

Chapter 8—Microbial Genetics
Note: For some really good animations concerning DNA replication, transcription and translation, go to
www.youtube.com and type: “DNA the secret of life PBS” along with “DNA replication” or “Transcription and
Translation” in the search window.
I.
Structure and Function of the Genetic Material.
a. Chromosome: An entire DNA molecule. Fig. 1.
b. Gene: Segment of DNA (or RNA in some viruses) that encodes a functional product, usually a
protein.
c. Genome: All of the genetic material in a cell/organism.
d. Genetics: Study of what genes are, how they carry information, how information is expressed,
how genes/DNA are replicated, and how they are passed from one generation to the next (or
passed between organisms).
e. Genomics: Molecular study of genomes.
f. Genotype and phenotype.
i. Genotype: Entire DNA complement of an organism/cell. (Combination of alleles).
ii. Phenotype: Physical expression of the genes. (Proteins/enzymes).
g. Flow of Genetic Information. Fig. 2.
i. The DNA of a cell replicates before cell division, so that each daughter cell gets an
identical copy of the chromosome.
ii. Genes contained on the DNA molecule can also be transcribed into mRNA and then
translated into a protein.
iii. Recombination.
h. DNA and Chromosomes.
i. DNA: Fig. 3.
1. Polymer of nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G).
2. Double helix.
3. "Backbone" is deoxyribose, phosphate, deoxyribose, phosphate…
4. Strands held together by hydrogen bonds between AT and CG (nitrogenous base
pairs).
5. Strands are antiparallel.
6. The entire length of the DNA double helix constitutes a single chromosome.
i. DNA Replication. Fig. 5
i. DNA helicase unwinds the double strand to begin the process of DNA replication.
1. This exposes a replication fork.
2. The original strand serves as the template for making a new strand.
ii. DNA is copied by DNA polymerase.
1. Initiated by an RNA primer.
2. The RNA primer is synthesized by RNA polymerase.
iii. The new DNA strand is synthesized in the 5  3 direction.
iv. Leading strand synthesized continuously. (Continuous strand).
v. Lagging strand synthesized discontinuously. (Discontinuous strand).
1. Okazaki fragments.
a. RNA primers of the lagging strand are removed by DNA polymerase, and
the Okazaki fragments are joined together by DNA ligase.
vi. DNA replication is semiconservative.
1. Each new DNA molecule contains a single parental strand and one new strand.
vii. Formation of new covalent bonds between nucleotides during DNA replication requires
an input of energy. Fig. 4
1. The nucleotides themselves provide the energy.
2. Nucleotides in their unbound form are really nucleoside triphosphates.
3. Cleaving the terminal pair of phosphates from the nucleoside triphosphate
provides the energy required to bind the nucleotides together.
viii. DNA replication in some bacteria like E. coli is bidirectional around the chromosome.
Fig. 6.
ix. DNA replication is very accurate; mistakes occur only once in every 1010 nucleotide
additions (1 in 10 billion) to a DNA molecule being synthesized. (There are about 3
billion nucleotides that make up the DNA found in a typical adult human cell).
1. DNA polymerase can excise any base that doesn’t properly complement the
parental DNA strand during synthesis and replace it with an appropriate base.
j. RNA and Protein Synthesis.
i. Transcription.
1. DNA is transcribed to make different forms of RNA (messenger RNA = mRNA,
transfer RNA = tRNA, and ribosomal RNA = rRNA).
2. RNA is a single-strand polymer of nucleotides: adenine (A), uracil (U), cytosine
(C), and guanine (G).
3. "Backbone" is ribose, phosphate, ribose, phosphate…
4. A strand of mRNA is synthesized using a single strand of DNA as a template.
a. Usually, only a single gene on the DNA strand is used as a template for
the synthesis of an mRNA molecule.
b. The process is similar to DNA replication, except for the fact that there are
no thymine (T) nucleotides in RNA; instead, the nucleotide uracil (U) is
present in place of thymine.
5. Transcription begins when RNA polymerase binds to the promoter sequence on
the DNA template strand. Fig. 7.
6. Synthesis of the RNA transcript proceeds in the 5  3 direction.
a. Example:
DNA coding (+) strand:
DNA template (-) strand:
mRNA transcript synthesized from DNA template:
5 ATGCAT 3
3 TACGTA 5
5 AUGCAU 3
7. RNA polymerase assembles the mRNA strand from free nucleotides through
complimentary base pairing using the DNA template strand as a guide.
8. Transcription stops when the RNA polymerase reaches the terminator sequence,
and the mRNA and the RNA polymerase are released from the DNA.
9. The process of transcription allows the cell to produce short-term copies of genes
that can be used as the direct source of information for protein synthesis.
a. Messenger RNA acts as an intermediate between the permanent storage
form, DNA, and the process that uses the genetic information, translation.
ii. Translation.
1. mRNA is translated in blocks called codons, each of which consist of 3
nucleotides. Fig. 8.
a. The sequence of codons specifies the order in which amino acids will be
assembled into proteins. (See chapter 2 for diagrams of the amino acids).
b. Often, multiple codons specify the same amino acid. This is referred to as
the degeneracy of the code.
i. There are only 20 amino acids, but there are 64 possible codons.
2. Translation of mRNA begins at the start codon AUG.
3. Translation ends at a STOP (nonsense) codon: UAA, UAG, UGA.
4. Translation is facilitated by the ribosome.
II.
5. tRNA molecules recognize the codons, and each carries a specific individual
amino acid. Fig. 9.
a. Each tRNA has a region called an anticodon that is complimentary to a
specific codon on the mRNA.
b. On the opposite end of the tRNA a single specific amino acid is attached.
c. The ribosome directs the orderly binding of tRNAs to the codons and
assembles the polypeptide chain.
d. The ribosome moves along the mRNA transcript in the 5  3 direction.
i. See Handout.
e. Multiple ribosomes can be translating the same mRNA molecule at the
same time.
f. In prokaryotes, translation can begin even before transcription is complete.
Fig. 10.
iii. RNA processing in Eukaryotes. Fig. 11.
1. Transcription occurs in the nucleus
2. The genes of eukaryotic DNA contain introns and exons.
a. Only exons code for proteins.
b. The mRNA transcript contains the same introns and exons as the DNA.
3. Small nuclear ribonucleoproteins (snRNPs) and ribozymes remove the introns
from the mRNA transcript and splice together the exons.
a. Ribozymes are like enzymes, but they are made of RNA and act on RNA.
i. Some introns ARE ribozymes…
4. RNA processing produces a functional mRNA molecule.
5. The mRNA leaves the nucleus for translation in the cytoplasm.
Regulation of Bacterial Gene Expression.
a. Cells conserve energy by synthesizing only the proteins they need at the time.
b. Constitutive genes (60-80% of all genes) are expressed at a fixed rate.
c. Other genes are expressed only as needed.
d. Repression and induction regulate mRNA transcription. Fig. 12.
i. Repression inhibits transcription of DNA.
1. Repressor proteins block RNA polymerase from initiating transcription of
particular genes.
2. This usually occurs in response to the over-abundance of some end-product
produced by a metabolic pathway.
ii. Induction.
1. Inducers turn on the transcription process of a gene.
e. The Operon Model of Gene Expression.
i. Described by Francois Jacob and Jacques Monod (1961).
ii. Describes the control of gene expression and protein synthesis by induction and
repression.
iii. Operon = A region of DNA in a bacterium where the genes responsible for coding for
proteins/enzymes that are involved in a single metabolic pathway are found together one
after the other.
iv. The lac operon, an inducible operon. Fig. 12 (step 1).
1. Involved in E. coli lactose metabolism.
2. Near the beginning of the lac operon is a regulatory gene (lac I) that codes for the
production of a repressor protein.
3. Within the operon are the following:
a. The control region, consisting of:
i. The promoter (where RNA polymerase starts transcription of the
operon).
III.
ii. The operator (where the repressor protein can bind under certain
conditions).
b. The structural genes, consisting of:
i. lac Z. The gene that codes for the production of Betagalactosidase (an enzyme) that cleaves lactose (a disaccharide) into
glucose and galactose (both monosaccharides).
ii. lac Y. The gene that codes for galactoside permease (lactose
transport protein) that brings lactose into the cell across the plasma
membrane.
iii. lac A. Codes for the enzyme transacetylase, which transfers acetyl
groups (CH3COO-) to Beta-galactoside sugars. This allows sugars
that can’t be metabolized to be exported from the cell instead of
accumulating to toxic levels within the cell.
c. RNA polymerase can be blocked by repressor protein when repressor
protein binds to the operator. This prevents transcription. Fig. 12 (step 2).
i. This occurs when lactose is absent.
ii. Repressor protein is reversibly bound to the operator.
d. When lactose is present, lactose is transported into the cell, and some
lactose is converted into the inducer allolactose. Fig. 12 (step 3).
i. Allolactose binds to the repressor protein, which undergoes a
conformational change. Therefore, the repressor protein can no
longer bind to the operator.
ii. RNA polymerase is now able to bind to the promoter and
transcribe the operon to produce an mRNA molecule, which is
then translated into the products above.
4. The presence/absence of lactose acts as an on/off switch for the entire lac operon.
a. When lactose is present, the operon is transcribed (induced), enabling the
lactose catabolic pathway to proceed.
b. When lactose is absent, the operon is repressed.
i. In this way, components of the lactose catabolic pathway are not
synthesized because they are not needed since there is no lactose
for them to work on.
5. Note: you will not be tested on: positive regulation of the lac operon,
epigenetic control, post-transcriptional control, and the tryptophan operon.
Mutation: Change in the Genetic Material.
a. Mutations that change the base sequence of DNA may be neutral (silent), beneficial (adaptive),
or harmful (deleterious).
i. Neutral mutations neither benefit nor damage the organism.
ii. Adaptive mutations could lead to antibiotic resistance or altered pathogenicity.
iii. Deleterious mutations lead to loss of function, the formation of a cancer, or death.
b. Types of Mutations.
i. Base substitution (point mutation) is where a single base is replaced with a different base.
Fig. 16.
1. If a base substitution results in an amino acid substitution in the synthesized
protein, this change in the DNA is known as a missense mutation. Fig. 17 a, b.
a. Example:
The cat saw the dog
The bat saw the dog
b. Note: Figs. 17 a and b in older versions of the textbook have a typo—the
“DNA coding strand” should be labeled as “DNA template strand”
instead.
IV.
ii. Nonsense mutation.
1. Creates a stop (nonsense) codon in the mRNA molecule that will prevent the
synthesis of a complete functional protein; only a fragment will be synthesized.
Fig. 17 c.
iii. Frameshift mutation.
1. One or a few nucleotide pairs are deleted or inserted in the DNA. Fig. 17 d.
2. This can shift the translational reading frame.
3. Example:
a. Before the frameshift: the fat cat ate the rat
b. After the frameshift: the atc ata tet her at
4. This type of mutation usually leads to a long stretch of new amino acids that
causes the protein to be non-functional.
c. Spontaneous mutations: Occur in the absence of a mutagen, and is due to a mistake in the DNA
copying process.
d. Mutagen: Agent that causes mutations.
e. Mutagens.
i. Chemical Mutagens.
ii. Radiation.
1. Ionizing radiation (X rays and gamma rays) causes the formation of ions and free
radicals that can react with nucleotides and the deoxyribose-phosphate backbone.
2. UV radiation causes bonds to form between adjacent pyrimidine bases, usually
thymines, forming thymine dimers. Fig. 20.
a. Light-repair enzymes can separate thymine dimers.
b. Nucleotide excision repairs mutations by cutting out and replacing the
damaged stretch of DNA. (This happens in humans during DNA
replication if a mistake is made).
f. The Frequency of Mutation.
i. Spontaneous mutation rate = 1 in 109 [1 billion] or 1 in 1010 [10 billion] replicated base
pairs or 1 in 106 [1 million] replicated genes, since the average gene has about 1000 base
pairs. (~3 billion nucleotides make up the DNA in a typical adult human cell).
ii. Mutagens increase the mutation rate to one in every 103-105 [1,000 – 100,000] replicated
genes.
g. Identifying Mutants.
i. Positive (direct) selection detects mutant cells because they grow or appear differently
from the rest of the population, which lacks the mutation. (Gain of function mutation).
1. When placed on a growth medium containing penicillin, penicillin resistant
bacteria grow while non-resistant bacteria die.
ii. Negative (indirect) selection detects mutant cells because they do not grow. (Loss of
function mutation).
1. Replica Plating. Fig. 21.
a. Mutant cells that have a nutritional requirement that is absent in the nonmutant parent cells are said to be auxotrophs.
h. Identifying Chemical Carcinogens.
i. Many mutagens are also carcinogenic (cancer causing).
ii. The Ames Test for Chemical Carcinogens. [DO NOT DISCUSS]
Genetic Transfer and Recombination.
a. Genetic recombination.
i. Exchange of genes between two DNA molecules to form new combinations of genes on a
chromosome.
1. Crossing over in eukaryotes. Fig. 23.
ii. Increases a population’s genetic diversity.
b.
c.
d.
e.
f.
g.
iii. Recombination is more likely to produce a beneficial outcome than mutation because
recombination will be less likely to destroy a gene’s function and may bring together a
combination of genes that enable the organism to carry out a valuable new function.
Vertical gene transfer.
i. When genes are passed from parent cell to daughter cell.
Horizontal gene transfer.
i. When genes are passed from one adult cell to another.
ii. The donor cell gives a portion of its DNA to a recipient cell.
1. Part of the donor’s DNA is incorporated into the recipient’s DNA, which is then
known as the recombinant cell.
iii. This is a very rare occurrence, and may happen in less than 1% of a population.
iv. Note: In this chapter, these processes apply specifically to bacteria, but horizontal gene
transfer in Eukaryotes via plasmids and transposons does occur.
Transformation in Bacteria. Fig. 25.
i. When a cell lyses, its DNA is released.
ii. Closely related living cells can take up fragments of that DNA and incorporate it into
their own DNA.
iii. This forms a hybrid recombinant cell.
iv. All descendants of the recombinant will be identical to it.
v. Transformation naturally occurs only among a few genera of bacteria: Bacillus,
Haemophilus, Neisseria, Acinetobacter, and some strains of Streptococcus and
Staphylococcus.
vi. Note: You will not be tested on Griffith’s experiments on transformation.
Conjugation in Bacteria. Fig. 26.
i. Involves a plasmid, which is a small, circular piece of DNA that replicates independently
from the cell’s chromosome.
1. Under normal conditions, the genes of a plasmid are usually not required for
growth.
ii. Requires direct cell-to-cell contact.
1. In G- species, plasmids carry genes that code for the synthesis of sex pili, which
help bring to two cells together for the transfer.
2. In G+ species, sticky surface molecules are synthesized that help the cells come
together for the transfer.
iii. The donor cell must carry the plasmid, and recipient cell usually does not.
iv. A single strand of the plasmid DNA is passed to the recipient cell.
v. Figs. 27 b and c show different forms of conjugation in E. coli.
Transduction in Bacteria. Fig. 28. [Discuss figure]
Plasmids and Transposons.
i. Genetic elements that provide additional mechanisms for genetic change.
ii. Occur in both prokaryotes and some eukaryotes (some yeasts).
iii. Plasmids.
1. Conjugative plasmid: Carries genes for sex pili and transfer of the plasmid.
2. Dissimilation plasmids: Encode enzymes for catabolism of unusual compounds.
(Petroleum, etc.).
3. Resistance (R) factors: Encode resistance to antibiotics, heavy metals, or cellular
toxins.
a. Treatment with antibiotics has selected for the survival of bacteria that
have R factors.
b. The transfer of resistance between bacterial cells of a population, and even
between bacteria of different genera, also contributes to the problem of
antibiotic resistant bacteria.
V.
iv. Transposons. Fig. 30.
1. Segments of DNA that can move from one region of a DNA molecule to another,
from one chromosome to another, or from a chromosome to a plasmid.
2. They occur in all organisms.
3. Contain insertion sequences for cutting and resealing DNA (transposase).
a. If insertion occurs within a gene, that gene will be rendered nonfunctional.
b. Frequency of transposition is about the same as the spontaneous mutation
rate of DNA in bacteria. Very rare. (Spontaneous mutation rate = 1 in 109
[1 billion] to 1 in 1010 [10 billion] replicated base pairs or 1 in 106 [1
million] replicated genes).
4. Complex transposons carry other genes in addition to the insertion sequences.
5. They can be carried between cells on plasmids or in viruses.
a. They can therefore be spread between members of the same species, and
between members of different species.
Genes and Evolution.
a. Genetic variation resulting from mutation, vertical gene transfer (crossing over), horizontal gene
transfer (transformation, conjugation, transduction, and transposition) drive evolution via natural
selection.