Microbial Genetics

Introduction to Genetics and Genes
Genetics: the study of the inheritance (heredity) of living things
Transmission of traits from parent to offspring
Expression and variation of those traits
The structure and function of the genetic material
How this material changes
Takes place on several levels: organismal, chromosomal, molecular

Microbial Genetics

The Nature of the Genetic Material
Must be able to self-replicate
Must be accurately duplicated and separated from each daughter cell

The Levels of Structure and Function of the Genome
Genome
Chromosome
Gene

Genome
The sum total of genetic material of a cell
Mostly in chromosomes
Can appear in nonchromosomal sites as well
In cells- exclusively DNA
In viruses- can be either DNA or RNA

Chromosome
A discrete cellular structure composed of a neatly packed DNA molecule
Eukaryotic chromosomes
DNA molecule tightly wound around histone proteins
Located in the nucleus
Vary in number from a few to hundreds
Can occur in pairs (diploid) or singles (haploid)
Appear linear
Bacterial chromosomes
Condensed and secured by means of histone-like proteins
Single, circular chromosome

Gene
A certain segment of DNA that contains the necessary code to make a protein or RNA molecule
Structural genes: code for proteins or code for RNA
Regulatory genes: control gene expression
Sum of all genes is an organism’s genotype
The expression of the genotype creates traits which make up the phenotype. Some genes may not be expressed in the phenotype.
All organisms contain more genes in their genotype than are manifested as a phenotype at a given time

The Size and Packaging of Genomes
Vary greatly in size
Smallest viruses- 4 or 5 genes
Escherichia coli- 4,288 genes
Human cell- 20,000 to 25,000 genes
The stretched-out DNA can be 1,000 times or more longer than the cell

The DNA Code: A Simple Yet Profound Message
1953: James Watson and Francis Crick
Discovered DNA is a gigantic molecule
A type of nucleic acid
With two strands combined into a double helix

General Structure of DNA
Basic unit: nucleotide
Phosphate
Deoxyribose sugar
Nitrogenous base

Nucleotides
Covalently bond to form a sugar-phosphate linkage- the backbone of each strand
Each sugar attaches to two phosphates
One bond is to the 5’ carbon on deoxyribose
The other is to the 3’ carbon

Nitrogenous Bases
Purines and pyrimidines
Attach by covalent bonds at the 1’ position of the sugar
Span the center of the molecule and pair with complementary bases from the other strands
The paired bases are joined by hydrogen bonds
Easily broken
Allow the molecule to be “unzipped”
Adenine always pairs with thymine
Guanine always pairs with cytosine

Antiparallel Arrangment
One side of the helix runs in the opposite direction of the other- antiparallel
One helix runs from 5’ to 3’ direction
The other runs from 3’ to 5’

The Significance of DNA Structure
Arrangement of nitrogenous bases
Maintains the code during reproduction (conservative replication of DNA)
Provides variety

Figure 9.5

DNA Replication: Preserving the Code and Passing it On
The process of the genetic code duplicated and passed on to each offspring
Must be completed during a single generation time

The Overall Replication Process
Requires the actions of 30 different enzymes
Separate the strands
Copy its template
Produce two new daughter molecules

Semiconservative Replication
Each daughter molecule is identical to the parent in composition, but only one strand is completely new
The parent DNA molecule uncoils
The hydrogen bonds between the base pairs are unzipped
Separates the two strands
Exposes the nucleotide sequence of each strand to serve as templates
Two new strands are synthesized by attachment of the correct complementary nucleotides to each single-stranded template

Refinements and Details of Replication
Origin of replication
Short sequence
Rich in A and T
Held together by only two H bonds rather than three
Less energy is required to separate the two strands
Helicases bind to the DNA at the origin
Untwist the helix
Break the hydrogen bonds
Results in two separate strands

DNA Polymerase III
Synthesizes a new daughter strand using the parental strand as a template
The process depends on several other enzymes as well, but key points about DNA polymerase III:
Nucleotides that need to be read by DNA polymerase III are buried in the double helix- so the DNA must first be unwound and the two strands separated
DNA polymerase III is unable to begin synthesizing a chain of nucleotides but can only continue to add nucleotides to an already existing chain
DNA polymerase III always reads the original strand from 3” to 5”
DNA polymerase III can only add nucleotides in one direction, so a new strand is always synthesized from 5’ to 3’

Elongation and Termination of the Daughter Molecules
As replication proceeds, the newly produced double strand loops down
DNA polymerase I removes RNA primers and replaces them with DNA
When the forks come full circle and meet, ligases move along the lagging strand
Begin initial linking of the fragments
Complete synthesis and separation of the two circular daughter molecules

Occasionally an incorrect base is added to the growing chain
Most are corrected
If not corrected, result in mutations
DNA polymerase III can detect incorrect, unmatching bases, excise them, and replace them with the correct base
DNA polymerase I can also proofread and repair

9.2 Applications of the DNA Code: Transcription and Translation
Central dogma
Genetic information flows from DNA to RNA to protein
The master code of DNA is used to synthesize an RNA molecule (transcription)
The information in the RNA is used to produce proteins (translation)
Exceptions: RNA viruses and retroviruses
Recently shown to be incomplete
In addition to the RNA that produces protein, other RNAs are used to regulate gene function
Many of the genetic malfunctions that cause human disease are found in these regulatory RNA segments

The Gene-Protein Connection
The Triplet Code and the Relationship to Proteins
Three consecutive bases on the DNA strand- called triplets
A gene differs from another in its composition of triplets
Each triplet represents a code for a particular amino acid
When the triplet code is transcribed and translated, it dictates the type and order of amino acids in a polypeptide chain
A protein’s primary structure determines its characteristic shape and function
Proteins ultimately determine phenotype
DNA is mainly a blueprint that tells the cell which kinds of proteins and RNAs to make and how to make them

The Major Participants in Transcription and Translation
Number of components participate, but most prominent:
mRNA
tRNA
regulatory RNAs
ribosomes
several types of enzymes
storehouse of raw materials
RNAs: Tools in the Cell’s Assembly Line
RNA differs from DNA
Single stranded molecule
Helical form
Contains uracil instead of thymine
The sugar is ribose
Many functional types, from small regulatory pieces to large structural ones
Only mRNA is translated into a protein molecule

Messenger RNA: Carrying DNA’s Message
A transcript of a structural gene or genes in the DNA
Synthesized by the enzyme RNA polymerase
Synthesized by a process similar to synthesis of the leading strand during DNA replication
The message of this transcribed strand is later read as a series of triplets (codons)

Transfer RNA: The Key to Translation
Also a copy of a specific region of DNA
It is uniform in length (75-95 nucleotides long)
Contains sequences of bases that form hydrogen bonds with complementary sections of the same tRNA strand
At these points the molecule bends back upon itself into several hairpin loops, giving the molecule a cloverleaf structure that then folds into a complex, 3-D helix

Transfer RNA: The Key to Translation cont.
Bottom loop of the cloverleaf exposes a triplet (the anticodon) that designates the specificity of the tRNA and complements mRNA’s codons
At the opposite end of the molecule is a binding site for the amino acid that is specific for that anticodon
For each of the 20 amino acids there is at least one specialized type of tRNA to carry it

The Ribosome: A Mobile Molecular Factory for Translation
The prokaryotic (70S) ribosome composed of tightly packed rRNA and protein
The interactions of proteins and rRNA create the two subunits of the ribosome that engage in final translation of the genetic code
The rRNA component of each subunit is a long polynucleotide molecule

Transcription: The First Stage of Gene Expression
Figure 9.11

Translation: The Second Stage of Gene Expression
All of the elements needed to synthesize a protein are brought together on the ribosomes
Five stages: initiation, elongation, termination, protein folding, and protein processing

Figure 9.12

Initiation of Translation
mRNA molecule leaves DNA transcription site
Is transported to ribosomes in the cytoplasm
Ribosomal subunits are specifically adapted to assembling and forming sites to hold the mRNA and tRNA’s
Prokaryotic ribosomes
70s size
50s subunit
30s subunit
Eukaryotic ribosomes
80s
60s subunit
40s subunit

The small subunit binds to the 5’ end of the mRNA
Large subunit supplies enzymes for making peptide bonds on the protein
The ribosome scans the mRNA by moving in the 5’ to 3’ direction along the mRNA
The first codon is the START codon (AUG but can rarely be GUG)
With the mRNA message in place on the ribosome, the tRNAs enter the ribosome with their amino acids
The complementary tRNA meets with the mRNA code
Guided by the two sites on the large subunit called the P site and the A site
The E site is where used tRNAs are released

The Master Genetic Code: The Message in Messenger RNA
The mRNA codons and the amino acids they specify
Redundancy of the genetic code: a particular amino acid can be coded for by more than a single codon
Wobble: in many cases, only the first two nucleotides are required to encode the correct amino acid- thought to permit some variation or mutation without altering the message

The Beginning of Protein Synthesis
Figure 9.15

The Termination of Protein Synthesis
Brought about by the presence of a termination codon: UAA, UAG, and UGA
Often called nonsense codons
Do not code for a tRNA
When reached, a special enzyme breaks the bond between the final tRNA and the finished polypeptide chain, releasing the polypeptide chain from the ribosome

Modifications to Proteins
Before it is released from the ribosome it starts to fold upon itself to achieve its biologically active tertiary conformation
Post-translational modifications may be necessary
Starting animo acid (methionine) clipped off
Cofactors added
Join with other proteins to form quaternary levels of structure

Transcription and Translation is Efficient
(Polyribosomes)

Eukaryotic Transcription and Translation: Similar Yet Different
Start codon is also AUG, but it codes for a different form of methionine
Eukaryotic mRNAs code for just one protein
The presence of the DNA in the nucleus means that eukaryotic transcription and translation cannot be simultaneous
mRNA in eukaryotes must pass through pores in the nuclear membrane and be carried to the ribosomes in the cytoplasm for translation

Most eukaryotic genes do not exist as an uninterrupted series of triplets coding for a protein
Introns- sequences of bases that do not code for protein
Exons- coding regions that will be translated into protein
Called a split gene- requires further processing before translation
Transcription of the entire gene with both exons and introns occurs first, producing a pre-mRNA

Most eukaryotic genes do not exist as an uninterrupted series of triplets coding for a protein
A series of adenosines is added to the mRNA molecule (protects it and directs it out of the nucleus)
A splicesome recognizes the exon-intron junctions and enzymatically cuts through them
The exons are joined end to end
Some introns do code for cell substances (in humans, introns represent 98% of the DNA)

Figure 9.17

The Genetics of Animal Viruses
Diverse
Some- nucleic acid is linear; others, circular
Most exist in a single molecule, but in a few it is in several
Most contain dsDNA or ssRNA, but other patterns exist
In all cases:
Viral nucleic acid penetrates the cell
The nucleic acid is introduced into the host’s gene-processing machinery
The virus instructs the host’s machinery to synthesize large numbers of new virus particles
Viral mRNA is translated into viral proteins on host cell ribosomes using host tRNA

9.3 Genetic Regulation of Protein Synthesis and Metabolism
Control mechanisms ensure that genes are active only when their products are required
Enzymes are produced as they are needed
Prevents the waste of energy and materials
Antisense RNAs, micro RNAs, and riboswitches provide regulation in prokaryotes and eukaryotes
Prokaryotes organize collections of genes into operons
Coordinated set of genes regulated as a single unit
Either inducible or repressible
Inducible- the operon is turned in by the substrate of the enzyme for which the structural genes code
Repressible- contain genes coding for anabolic enzymes; several genes in a series are turned off by the product synthesized by the enzyme

The Lactose Operon: A Model for Inducible Gene Regulation in Bacteria
Best understood cell system for explaining control through genetic induction
Lactose (lac) operon
Regulates lactose metabolism in Escherichia coli
Three important features:
The regulator (a gene that codes for a protein capable of repressing the operon [a repressor])
The control locus
Promoter- recognized by RNA polymerase
Operator- a sequence that acts as an on/off switch for transcription
The structural locus, made up of three genes each coding for a different enzyme needed to catabolize lactose

Figure 9.18

A Repressible Operon
Normally the operon is in the “on” mode and will be turned “off” only when the nutrient is no longer required
The excess nutrient serves as a corepressor needed to block the action of the operon
Example, arg operon

Figure 9.19

Antibiotics that Affect Transcription and Translation
Some infection therapy is based on the concept that certain drugs react with DNA, RNA, or ribosomes and alter genetic expression
Based on the premise that growth of the infectious agent will be inhibited by blocking its protein-synthesizing machinery selectively
Drugs that inhibit protein synthesis exert their influence on transcription or translation
Antibiotics often target the ribosome- inhibiting ribosomal function and ultimately protein synthesis

Mutations: Changes in the Genetic Code
Genetic change is the driving force of evolution
Mutation: when phenotypic changes are due to changes in the genotype
An alteration in the nitrogen base sequence of DNA
Wild type: a microorganism that exhibits a natural, nonmutated characteristic
Mutant strain: when a microorganism bears a mutation
Useful for tracking genetic events,
Unraveling genetic organization, and
Pinpointing genetic markers

Causes of Mutations
Spontaneous mutation: random change in the DNA arising from errors in replication
Induced mutation: results from exposure to known mutagens

Categories of Mutations
Point mutations: involve addition, deletion, or substitution of single bases
Missense mutation: any change in the code that leads to placement of a different amino acid
Can create a faulty, nonfunctional protein
Can produce a protein that functions in a different manner
Can cause no significant alteration inI protein function
Nonsense mutation: changes a normal codon into a stop codon
Silent mutation: alters a base but does not change the amino acid and thus has no effect
Back-mutation: when a gene that has undergone mutation reverses to its original base composition

Categories of Mutations cont.
Frame shift mutations:
mutations that occur when one or more bases are inserted into or deleted from a newly synthesized DNA strand
Changes the reading frame of the mRNA
Nearly always result in a nonfunctional protein

Repair of Mutations
Most ordinary DNA damage is resolved by enzymatic systems specialized for finding and fixing such defects
DNA that has been damaged by UV radiation
Restored by photoactivation or light repair
DNA photolayse- light-sensitive enzyme
Excision repair
Excise mutations by a series of enzymes
Remove incorrect bases and add correct one

The Ames Test
Rapid screening system
Detects chemicals with carcinogenic potential
Any chemical capable of mutating bacterial DNA can similarly mutate mammalian DNA

Figure 9.22

Positive and Negative Effects of Mutations
Mutations are permanent and inheritable
Most are harmful but some provide adaptive advantages

DNA Recombination Events
Recombination: when one organism donates DNA to another organism
The end result is a new strain different from both the donor and the original recipient
Bacterial plasmids and gene exchange
Recombinant organism: Any organism that contains (and expresses) genes that originated in another organism

Transmission of Genetic Material in Bacteria
Usually involves small pieces of DNA (plasmids or chromosomal fragments)
Plasmids can replicate independently of the bacterial chromosome
Chromosomal fragments must integrate themselves into the bacterial chromosome in order to replicate
Three means of genetic recombination in bacteria
Conjugation
Transformation
Transduction

Conjugation: Bacterial “Sex”

Biomedical Importance of Conjugation
Resistance (R) plasmids, or factors- bear genes for resisting antibiotics
Can confer multiple resistance to antibiotics to a strain of bacteria
R factors can also carry resistance to heavy metals or for synthesizing virulence factors

Transformation: Capturing DNA from Solution

Griffith demonstrated that DNA released from a killed cell can be acquired by a live cell
Later studies supported this
Nonspecific acceptance by a bacterial cell- transformation
Facilitated by special DNA-binding proteins on the cell wall
Competent cells- capable of accepting genetic material
Useful for certain types of recombinant DNA technology

Transduction: The Case of the Piggyback DNA

Transposons: “This Gene is Jumpin”