Microbial Genetics Methanococcus jannaschii http://www.tigr.org/CMR2/BackGround/arg.html

Chapter 9 Topics - Genetics - Flow of Genetics - Regulation - Mutation - Recombination

The sum total of genetic material of a cell is referred to as the genome. Fig. 9.2 The general location and forms of the genome

Figure 7.2 Bacterial genome-overview

Chromosome Prokaryotic Eukaryotic Histonelike proteins condense DNA Eukaryotic Histone proteins condense DNA Subdivided into basic informational packets called genes

Genes Three categories Genotype Phenotype Structural Regulatory Encode for RNA Genotype sum of all gene types Phenotype Expression of the genotypes

Introduction A. Gene – a segment of DNA that codes for a protein. B. Genetics – a study of genes or heredity, how traits are passed from one generation to the next. C. Microbe traits that are heritable 1. Morphology – cell shape and arrangement. 2. Structures – capsules, flagella, endospores, cell wall type, etc. 3. Biochemistry – Enzymes, metabolic pathways etc.

The prokaryotic chromosome Only one chromosome, only one copy of the genotype. Doesn’t deal well with deleterious mutations. Makes up for this with bacterial numbers and mutability.

Roles of DNA A. Replication B. Gene expression cell division need accurate copy B. Gene expression DNA RNA Protein Figure 6.2

Structure of DNA A. Two strands B. Nucleotides C. Double helix 1. Hydrogen bonds between strands 2. Neighboring deoxyribose connected a. 3’ of one deoxyribose to 5’ of next deoxyribose b. Phosphate in between C. Double helix D. Complimentary base pairing G and C A and T Antiparallel strands Figure 6.1

Structure Nucleotide Double stranded helix Phosphate Deoxyribose sugar Nitrogenous base Double stranded helix Antiparallel arrangement

Nitrogenous bases Purines Adenine Guanine Pyrimidines Thymine Cytosine

DNA Replication A. Template Concept Each side of a DNA molecule is a template for the synthesis of a new strand due to complementary base pairing

Semi Conservative Replication Each half of the original strand is incorporated into the new double strand.

Events of replication 1. Origin/s a. In prokaryotes = 1 b. In eukaryotes = many 2. Strand separation a. Helicase- opens the strands b. Single strand binding proteins – keeps strands separated c. Topoisomerases – keep DNA strands from tangling up. 3. Building new strands a. DNA polymerase I and III – adds in new nucleotides b. RNA primase – makes a short RNA primer on the lagging strand c. DNA ligase – binds Okazaki fragments

Enzymes Helicase DNA polymerase III Primase DNA polymerase I Ligase Gyrase

The function of important enzymes involved in DNA replication. Table 9.1 Some enzymes involved in DNA replication

topoisomerases

Bacterial chromosomes Replication of circular chromosome 1. Origin of replication a. bubble forms b. DNA unwinds 2. Replication occurs in both directions a. Two replication forks b. Continues until replication forks meet 3. Strands separate Figure 6.4

Fig. 9.6

Fig. 9.6a

Fig. 9.6b

Fig. 9.6c

Fig. 9.6d

Figure 7.8 The central dogma of genetics 5´ 3´ DNA (genotype) 3´ 5´ Transcription mRNA 5´ 3´ Translation by ribosomes NH2 Methionine Arginine Tyrosine Leucine Polypeptide Phenotype

RNA Transcription Codon Message RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) Codon

Transcription A single strand of RNA is transcribed from a template strand of DNA RNA polymerase catalyzes the reaction Synthesis in 5’ to 3’ direction

mRNA Copy of a structural gene or genes of DNA Can encode for multiple proteins on one message Thymidine is replaced by uracil The message contains a codon (three bases)

The synthesis of mRNA from DNA. Fig. 9.12 The major events in mRNA synthesis

tRNA Copy of specific regions of DNA Complimentary sequences form hairpin loops Amino acid attachment site Anticodon Participates in translation (protein synthesis)

Important structural characteristics for tRNA and mRNA. Fig. 9.11 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. Fig. 9.9 Summary of the flow of genetics

Codons Triplet code that specifies a given amino acid Multiple codes for one amino acid 20 amino acids Start codon Stop codons

The codons from mRNA specify a given amino acid. Fig. 9.14 The Genetic code

Protein Translation Protein synthesis have the following participants mRNA tRNA with attached amino acid Ribosome

Participants involved in the translation process. Fig. 9.13 The “players” in translation

Figure 7.15 Prokaryotic ribosomes-overview

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

For procaryotes, translation can occur at multiple sites on the mRNA while the message is still being transcribed. Fig. 9.17 Speeding up the protein assembly line in bacteria

Figure 7.19 A polyribosome in a prokaryotic cell-overview

Regulation Lactose operon Repressible operon Antimicrobials sugar Amino acids, nucleotides Antimicrobials

Regulation of Genetic Expression Nature of prokaryotic operons An operon consists of a promoter and a series of genes Some operons are controlled by a regulatory element called an operator © 2012 Pearson Education Inc.

Figure 7.20 An operon Operon Promoter Operator Structural genes Regulatory gene 1 2 3 4 3´ 5´ Template DNA strand

Regulation of Genetic Expression Nature of prokaryotic operons Inducible operons must be activated by inducers Lactose operon Repressible operons are transcribed continually until deactivated by repressors Arginine operon © 2012 Pearson Education Inc.

The regulation of sugar metabolism such as lactose involves repression in the absence of lactose, and induction when lactose is present. Fig. 9.19 The lactose operon in bacteria

The regulation of amino acids such as arginine involves repression when arginine accumulates, and no repression when arginine is being used. Fig. 9.20 Repressible operon

Antimicrobials Ex. Antibiotics and drugs can inhibit the enzymes involved in transcription and translation

Mutations Changes made to the DNA 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

Mutations A. Mutation – any change in the DNA base sequence. B. May be harmless, harmful, or beneficial C. can only be passed to next generation if mutation occurs in sex cells, not somatic cells.

Causes 1. Spontaneous – errors in replication 2. Radiation (Sun, X-rays, nuclear bombs) 3. Chemicals (nicotine, formaldehyde)

Types of mutations 1. Point Mutations – addition, deletion, substitution of a single base – One nitrogenous base is changed. May not be so severe since only one base, but can be severe. EX. Sickle cell anemia. 2. Missense Mutation – base changes cause a different amino acid to be inserted and a protein is made but an incorrect one 3. Nonsense Mutation – base changes result in normal codon change into a stop codon so no protein is made due to an early stop signal 4. Neutral or Silent Mutations – no effect on protein product (base sequence is read differently, but still same protein sequence)

5. Frameshift mutation Insertion – one or more nucleotides is added to the sequence. Deletion- one or more nucleotides is lost in the sequence. Frame-shift mutations are very severe because it throws off the entire reading frame of a gene. THE FAT CAT ATE THE BIG RAT THE FAC ATA TET HEB IGR AT

Effects of mutations Positive effects for the cell Allow cells to adapt Negative effects for the cell Loss of function Cells cannot survive

Ways bacteria acquire DNA information Transformation – picking up DNA from the environment. 2. Conjugation – primitive “mating” in between bacteria 3. Transduction – Bacterial DNA carried from one bacteria to another by a bacteriophage.

1. Transformation a. DNA exits one cell, taken up by another cell 1. Natural 2. few bacteria take up DNA fragments b. Artificial--induced in laboratory 1. useful tool for recombinant DNA technology Figure 6.20

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. Fig. 9.25 Griffith’s classic experiment in transformation

2. Conjugation a. Conjugative plasmids sex pilus connect two cells Transfers plasmid one cell F+ one cell F- Figure 6.21

Conjugation cont. 3. linear strand is copied forming a complete plasmid 4. both cells are now F+ Figure 6.21

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. Fig. 9.24 Conjugation: genetic transmission through direct contact

3. Transduction a. Bacteriophage 1. virus that infects bacteria 2. reproduce in bacteria 3. some phages contain bacterial DNA rare event transducing particle 4. cell lysis and release normal phage transducing particles Figure 6.23

Transduction 5. Transducing particles 6. genetic exchange infect other bacteria inject bacterial DNA into new cell 6. genetic exchange one bacteria cell to another 7. integration into chromosome Figure 6.23

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. Fig. 9.26 Generalized transduction

Genetic transfer based on specialized transduction. Fig. 9.27 Specialized transduction