Chapter 8 - Microbial Genetics

Gene expression

• genetic information is used within a cell to produce the proteins needed for the cell to function

Gene Recombination

Genetic information can be transferred between cells of the same generation.

DNA replication 

Genetic information can be transferred between generations of cells.

Genetics

• The study of genes, how they carry information, how information is expressed and how genes are replicated

Chromosomes

structures containing DNA that physically carry hereditary information, the chromosomes contain genes

Genes

segments of DNA that encode functional products, usually proteins

Genome 

all the genetic information in a cell 

Genetic Code

• A set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

Central Dogma

• DNA → RNA → protein

Transcription

DNA → mRNA

Gene expression

• A gene is expressed when we have the protein product

• In microbes, most proteins are either enzymatic or structural

Transcription

• DNA → mRNA

• scribe → copy

• staying in the same language of nucleic acid

DNA

• deoxyribonucleic acid (DNA)

  • Hydrogen bonds between the bases

  • strands are complementary

  • uniform width

  • antiparallel

  • order of the nitrogen-containing bases forms the genetic instructions of the organism

RNA

• single stranded 

• many different types 

• 5-carbon ribose sugar

• extra OH group

• contains uracil instead of thymine 

Transcription - general

• synthesis of a complementary mRNA strand from a DNA template

• transcription begins when RNA polymerase binds to the promoter sequence on DNA

  • does not transcribed

• proceeds in the 5’-3’ direction; only 1 of the two DNA strands is transcribed

• transcription stops when it reaches the terminator sequence on DNA

3 stages of transcription

• Initiation: RNA polymerase binds to a promoter

• Elongation: Synthesis by adding complementary nucleotides

• Termination: RNA polymerase reaches the terminator

Transcription

• RNA polymerase bind to the promoter, and DNA unwinds at the beginning of a gene

• RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA

• the site of synthesis moves along DNA; DNA that has been transcribed rewinds

• Transcription reaches the terminator

• RNA and RNA polymerase are released and the DNA helix re-forms.

• the strand that the mRNA uses → template strand as the mRNA uses that strand as a template and makes bases complimentary to it

• the other strand is called the coding strand because it has the same nucleotides as the mRNA except thymine instead of uracil

Transcription - Eukaryotic Specific

• transcription occurs in the nucleus whereas translation occurs in the cytoplasm

• Exons are regions of DNA that code for proteins

• Introns are regions of DNA that do not code for proteins 

• Spliceosome → a large RNA-protein complex that removes introns and splices together exons 

  • composed of small nuclear ribonucleoproteins (snRNA) and RNA

RNA processing

In the nucleus, a gene composed of exons and introns is transcribed to RNA by RNA polymerase

Processing involves snRNPs in the nucleus to remove the intron-derived RNA and splice together the exon-derived RNA into mRNA

After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis.

Translation direction

• mRNA → protein → translate between two languages (nucleic acid to amino acid)

The components required for protein synthesis

• messenger RNA (mRNA): DNA → info → ribosomes

• Ribosome

  • ribosomal RNA (rRNA): integral part of ribosomes

  • Ribosomal proteins 

• Amino acids

  • Transfer RNA (tRNA): transports amino acids during protein synthesis

Translation

• mRNA is translated into the “language” of proteins 

• Codons are groups of 3 mRNA nucleotides that code for a particular amino acid 

• translation of mRNA begins at the start codon: AUG

• Translation ends at nonsense codons: UAA, UAG, and UGA

• Codons of mRNA are “read” sequentially

• tRNA molecules transport the required amino acids to the ribosome

• tRNA molecules also have an anticodon that base-pairs with the codon

• amino acids are joined by peptide bonds

• In bacteria, translation can begin before transcription is complete

The genetic code

• 61 sense codons encode the 20 amino acids

• The genetic code involves degeneracy, meaning each amino acids is coded by several codons

The regulation of bacterial gene expression 

• constitutive genes are expressed at a fixed rate

• other genes are expressed only as needed 

  • inducible genes

  • repressible genes

Pre-transcriptional control → Induction

• An inducible operon includes genes that are in the “off” mode with the repressor bound to the DNA, and is turned “on” by the environmental inducer. 

  • When turned “on”, induction turns on gene expression

  • initiated by an inducer, which binds to the repressor, turning it inactive

  • The default position of an inducible gene is off

Pre-transcriptional control - repression

• Repression inhibits gene expression and decreases enzyme synthesis

  • mediated by repressors, proteins that block transcription

• Repressible operon default: “on” mode → meaning the DNA gene is being expressed because the repressor is inactive

  • turned “off” by the environmental corepressor and repressor.

Operon Model of Gene Expression

• Promoter: segment of DNA where RNA polymerase initates (or promotes) transcription of structural genes

• Operator: segment of DNA that controls transcription of structural genes

• Operon: set of operator and promoter sites and the structural genes they control

• unique to prokaryotes 

• In an inducible operon, structural genes are not transcribed unless an inducer is present 

  • E.coli → enzymes of the lac operon are needed to metabolize lactose

  • In the absence of lactose, → repressor binds to the operator, preventing transcription

  • in the presence of lactose, the metabolite of lactose (allolactose → inducer) binds to the repressor

    • The repressor cannot bind to the operator, and transcription occurs

• In repressible operons, structural genes are transcribed until they are turned off

  • Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis 

Lac operon - an inducible operon 

• Structure of the operon

  • promoter → operator → ZYA structural genes

  • operon is regulated by the product of the reg. gene (gene before promoter)

Lac operon → Repressor active

• I gene is transcribed and translated to make a repressor protein

  • transcription → makes repressor mRNA

• translation → makes active repressor protein

• The active repressor protein binds to the operator region of the operon

• When the repressor is bound to the operator → RNA polymerase can’t move forward to transcribe the structural genes (Y,Z, and A)

• as a result → transcription is blocked and the genes that normally make the enzymes 

Structure of the Lac Operon

• The lac operon controls the breakdown of lactose in E. coli 

  • includes 3 main region:

    • regulatory gene: makes the repressor protein, which blocks transcription 

    • control region: contains promoter and operator 

      • The promoter is where RNA polymerase binds to start transcription 

      • The operator is the “switch” that the repressor binds to, turning the operon off

    • Structural Genes (ZYA): code for enzymes that break down lactose 

      • lacZ: makes β-galactosidase (breaks lactose into glucose + galactose) 

      • lacY: makes permease (helps lactose enter the cell) 

      • lacA: makes transacetylase (detoxifies byproducts) 

• When the inducer (allolactose) binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately leading to the production of the enzymes required for lactose catabolism. 

Trp operon - A Repressible Operon 

• The operon consists of the promoter and operator and structural genes that code for the protein 

• the operon is regulated by the product of the regulatory gene

• The Trp operon controls the production of enzymes needed to make the amino acid tryptophan 

• The regulatory gene → repressor mRNA → inactive repressor protein (cannot bind to the operator region) → allows RNA polymerase to attach to the promoter and trancribe the structural genes → resulting mRNA is then translated into enzymes that synthesize tryptophan

• the cell produces tryptophan when its levels are low 

When tryptophan levels are high:

• the amino acids acts as a corepressor

• tryptophan binds to the inactive repressor protein → changing its shape and activating it

• the active repressor then binds to the operator region of the DNA

  • blocks RNA polymerase from binding to the promoter or moving forward → transcription stops

  • no mRNA or enzymes are made

Summary: Repressor active → operon off.

The operon shuts down when enough tryptophan is present — a negative feedback loop that prevents waste of energy and resources.

Changes in genetic material 

Mutation: a permanent change in the base sequence of DNA 

• mutations may be neutral, beneficial or harmful

Mutagens are agents that cause mutations

Spontaneous Mutations occur in the absence of a mutagen

Types of mutations

• Base Substitution

  • point mutation

  • change in 1 base of DNA

  • can be deleterious or result in no change

  • CG → AT

• Missense Mutation

  • Base substitution results in a change in an amino acid

• Nonsense Mutation

  • base substitution results in a nonsense (stop) codon

  • The stop codon is premature, resulting in a truncated protein product '

• Frameshift mutation

  • insertion or deletion of one or more nucleotide pairs

  • shifts the translational reading frame

Chemical Mutagens

• increase the mutation rate

• Nitrous Acid: causes A to bind with C instead of T

• Nucleoside analog: incorporates into DNA in place of a normal base, causes mistakes in base pairing

  • antiviral medications

• Frameshift mutagens

Radiation

• ionizing radiation (x-rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the sugar-phosphate backbone of DNA

• non-ionizing radiation (UV light) produces thymine dimers

Photolyases

• enzymes that are found in some microorganisms that use light energy to break thymine dimers

  • Say if A pairs with G instead of T, doing so → the DNA strand would be cut along with the neighbors surrounding the mispair → the missing patch would be replaced with correct nucleotides → DNA ligase seals the gap

Frequency of mutations

• spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes

  • The mutation rate is dependent on the organism in question

• Mutations occur randomly along the genome

• Mutagens increase the mutation rate by 10-5 or 10-3 replicated gene

Identifying Mutants

• Positive (direct) selection detects mutant cells because they grow or appear different than unmutated cells

• Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function

  • use of replica plating 

• Auxotroph: mutation that has a nutritional requirement that was absent in the parent 

Replica Plating process

• A sterile velvet surface is pressed on the grown colonies in a master plate

• Cells from each colony are transferred from the velvet to NEW plates (one with histidine and the other one without)

• plates are incubated

• Growth on plates is compared. A colony that grows on the medium with histidine but cannot grow on the medium without histidine is auxotrophic for histidine

Ames test

• exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation

  • indicates the degree to which a substance is mutagenic

• 2 cultures of Salmonella that lost the ability to synthesize histidine are prepared

• The suspected chemical mutagen is added to the experimental sample only, and the control stays without it, but rat liver is added to both

• Each sample is poured into a medium that does not contain histidine → Incubation → Only bacteria that were histidine dependent reverted into having histidine colonies. → shown to be more on the plate that had the suspect mutagen than the one that did not

• The higher the concentration of mutagen used, the more revertant colonies are obtained

DNA and chromosomes

• eukaryotic DNA is linear and segmented

• Bacteria usually have a single circular chromosome made of DNA and associated proteins

DNA replication - ORI

• The origin of replication is where replication begins

• One strand serves as a template for the production of the second strand

  • semiconservative replication 

DNA replication of a plasmid

• Most bacterial DNA replication is bidirectional 

• Each offspring cell receives one complete copy of the DNA molecule 

• Replication is highly accurate due to the proofreading capability of DNA polymerase 

• Mutation: 1 in every 10 billion base incorporate

Enzymes in DNA replication

• DNA gyrase: relaxes supercoiling ahead of the replication fork

• DNA ligase: makes covalent bonds to join DNA strands, Okazaki fragments and new segments in excision repair

• DNA polymerases: synthesize DNA, proofread and facilitate repair of DNA

• Helicase: unwinds double stranded DNA

• Methylase: adds methyl group to selected bases in newly made DNA

• Primase: an RNA polymerase that makes RNA primers from a DNA template

• Topoisomerase or gyrase: relaxes supercoilng ahead of the replication fork; separates DNA circles at the end of the DNA replication

DNA replication process

• Topoisomerase and gyrase relax the strands

  • targeted by quinolone antibiotics

• helicase separates the strands

• A replication fork is created

• Primase lays down the RNA primer of which the new strand is synthesized

• DNA polymerase adds nucleotides to the DNA strand in the 5 ‘ to 3 ‘ direction

  • The leading strand is synthesized continuously (5 prime to 3 prime), and the lagging strand is synthesized discontinuously w Okazaki fragments

  • DNA polymerase removes RNA primers, Okazaki fragments are joined with the help of the DNA polymerase and ligase

Genetic Transfer

• Horizontal gene transfer: transfer of genes between cells of the same generation

  • only for prokaryotes

• Vertical gene transfer: transfer of genes from an organism to its offspring

Mobile genetic elements

• move from 1 chromosome to another

  • transposons (both prokaryotes and eukaryotes)

• Or move from 1 cell to another

  • plasmids (prokaryotes only)

Plamids 

• self-replicating circular pieces of DNA 

• 1 to 5% of the size of a bacterial chromosome 

• Often code of proteins that enhance the pathogenicity of a bacterium 

• Conjugative plasmid: 

  • Type of plasmid that can move from one cell to another through carrying transfer or tra genes that code for proteins intended to carry a sex pilus → bridge like structure between cells 

• Dissimilation plasmids 

  • encode enzymes/genes for the breakdown or to metabolize unusual organic compounds such as pesticides, hydrocarbons or aromatic compounds 

• Resistance factors 

  • encode antibotic resistance

Transposons 

• segments of DNA that can move from 1 region of DNA to another 

• Enzyme recognizes and binds to the inverted repeat sequences at both ends of the transposon 

• Enzymes and transposons form a looped structure called the transposition complex

• The enzyme cut the transposon out of the donor DNA molecule

• It finds a new target site somewhere else in the DNA

• It is inserted into a new location → joining the DNA sequence with the IR at both ends

Horizontal gene transfer in bacteria

• conjugation

  • Bacterial plasmids can be transferred from 1 cell to another via a sex pilus or a bridge

  • if F+ goes to F-, F- cell becomes F+

• Transformation

  • genes are transferred from 1 bacterium to another as “naked” DNA

  • the “naked” DNA can be incorporated into the bacterial genome

  • for stable integration into the bacterial genome, crossing over must occur

  • review Griffith’s experiment

• Transduction

  • DNA is transferred from 1 donor cell to a recipient via a bacteriophage

  • Generalized transduction: random bacterial DNA is packaged inside a phage and transferred to a recipient cell

  • A phage infects the donor bacterial cell

  • phage DNA and proteins are made, and the bacterial chromosome is broken into pieces

  • pieces of bacterial DNA are packaged into a phage capsid

  • donor cell lyses and releases phage particles containing bacterial DNA

  • phage infects the recipient cell

  • Inside the recipient cell, the donor bacterial DNA can combine (recombine) with the recipient’s own DNA.

    This creates a recombinant cell, meaning its genome is now a mix of both the donor and recipient bacteria.