Microbial Genetics

central dogma flow of genetic information

DNA → RNA → protein

Explain supercoiling.

1. One part of circle is laid over the other.

2. Helix makes contact in two places.

3. DNA gyrase breaks double-strand.

4. Unbroken helix is passed through the break.

5. The break is resealed by DNA gyrase.

DNA gyrase

• topoisomerase

inserts and removes supercoils

______ is one of the mechanisms to introduce diversity and boosts survival in hostile environments.

horizontal gene transfer

______ permits new mutations to be quickly generated and stabilized in the progeny, improving the overall genetic pool.

fast cell cycles

major components of DNA structure

1. sugar-phosphate backbone

   • DNA nucleotides linked by phosphodiester bonds

2. nucleotides held together by hydrogen bonds

3. sugar-phosphate backbone of one strand is antiparallel

4. complementary base pairing (A=T/C=G)

features of replication

1. semiconservative replication creates complementary strands

   • one parental strand (template) + one newly synthesized strand

2. bidirectional

   • replication moves along in both directions from starting point

Replication starts at the ____.

origin of replication (Ori)

• specific sequences of DNA that are recognized by specific proteins

How many origins of replication are in prokaryotes?

one

How many origins of replication are in eukaryotes?

multiple

Replication is done by which enzyme?

DNA polymerase

DNA helicase

unwinds the DNA forming a replication bubble

• each bubble = 2 replication forks

unwinds double stranded DNA

DNA gyrase

relaxes supercoiling ahead of the replication fork

• SPECIFIC TO BACTERIA

DNA ligase

binding of nicks in DNA during synthesis and repair

DNA Polymerase I

removes RNA primers, replaces gaps between Okazaki fragments

DNA Polymerase III

1. adds bases to new DNA chain at the 3’ OH

2. proofreading

   • only goes 5’-3’

primase

RNA polymerase that synthesizes an RNA primer from a DNA template

codons

base sequence of the mRNA molecule read in groups of 3 bases

• specific amino acid sequence of the polypeptide

anticodon

three nucleotides by which a tRNA recognizes an mRNA codon

transcription

DNA → RNA

enzyme for transcription

RNA polymerase

Differentiate between DNA and RNA.

1. DNA contains deoxyribose sugar, while RNA contains ribose.

2. DNA contains thymidine, while RNA contains uracil.

three main forms of RNA produced by the RNA polymerase from the DNA during transcription

1. messenger RNA (mRNA)

2. transfer RNA (tRNA)

3. ribosome RNA (rRNA)

mRNA

has the code for proteins

tRNA

1. reads the codon and interprets the nucleic acid sequence into an anticodon

2. carries amino acids to the ribosome

rRNA

two distinct ribosome pieces of different sizes

• small and large subunit of ribosome

Where do transcription and translation occur in prokaryotes?

in the cytoplasm simultaneously

Where do replication, transcription, and translation occur in eukaryotes?

1. Replication and transcription → nucleus

2. Translation → cytoplasm

three stages of transcription

1. initiation

2. elongation

3. termination

initiation

1. occurs at the promotor

2. initiated by RNA polymerase

3. synthesizes mRNA

promotor

specific DNA sequences upstream of the gene

eukaryotic promotor

TATA box

elongation

• transcription

1. RNA polymerase unwinds the DNA

2. reads the DNA sequence 5’-3

3. transcribes mRNA sequence

termination

• transcription

1. RNA polymerase falls off

2. transcription ends at this DNA sequence

translation

mRNA → protein

The tRNA anticodon is _____ to the mRNA codon.

complementary

• binds the mRNA in the ribosome

Where does translation occur?

surface of the ribosomes

prokaryotic ribosomal units

30S and 50S = 70S

eukaryotic ribosomal units

40S and 60S = 80S

translation

mRNA → protein

steps of translation

1. initiation

   • mRNA binds to the small subunit

   • tRNA anticodon binds to mRNA at the start codon

2. elongation

   • addition of amino acids to the polypeptide chain

3. termination

   • elongation stops at the stop codon

   • ribosomal subunits separate

   • polypeptide is released

initiator tRNA in eukaryotes

methionine

initiator tRNA in prokaryotes

formyl methionine

differences between eukaryotic and prokaryotic transcription and translation

1. In eukaryotes, transcription and translation do not occur simultaneously, but in prokaryotes they do.

2. mRNA in eukaryotes code for a single protein, but mRNA in prokaryotes encode many proteins.

3. Eukaryotes have introns, but prokaryotes do not.

Microbes regulate protein function in 2 ways:

1. control the amount of an enzyme or protein

2. control the activity of an enzyme or protein

constitutive genes

essential gene that is expressed at a fixed rate

• always turned on

adaptive genes

expressed on an as needed basis

inducible genes

OFF and requires a signal to be turned on

repressible genes

ON and requires a signal to be turned off

Operons are controlled by:

regulon

regulon

single regulatory protein that controls more than one operon

essential steps in component signal transduction system

1. monitor environment

2. transmit signal to a target to trigger change

quorum sensing

1. bacterial communication via small peptides or nonpeptide organic molecules

   • can lead to swarming, attachment/detachment to or from surfaces

   • important for virulence and survival

2. requires a quorum

quorum

sufficient bacteria and signals to trigger a certain adaptation

quorum sensing when there is no quorum (too few bacteria)

1. internal level of autoinducer = low

2. no change in transcription

quorum sensing when quorum is reached (high numbers of bacteria)

1. internal level of autoinducer = high

2. gene transcription triggered

operon

two or more genes transcribed into a single RNA

• under the control of a single regulatory site (operator and promotor)

polycistronic mRNA synthesis

when more than one gene is being produced

Inducible genes are influenced by:

substrates

lac operon

entire stretch of DNA that includes the promotor, operator, and the structural genes that it controls

lactose operon

produces an enzyme to break down lactose when lactose is present

Z gene

structural gene for β-galactosidase

• breaks down lactose

Y gene

structural gene for β-galactosidase permease

• allows lactose to enter the cell easier

A gene

structural gene for β-galactosidase transacetylase

• keeps lactose in cell

I gene

codes for a repressor protein (regulatory gene) that can block the polymerase from acting by binding to the operator

• NOT PART OF OPERON

repression proteins

regulatory protein that binds to specific sites on DNA and blocks transcription

• always on

• alters protein production

plasmids

an extra chromosomal DNA molecule that independently replicates DNA (replicons)

• not essential

replicon

genetic component that possesses all the necessary elements to self replicate (ori)

episome

a plasmid with the ability to integrate into the chromosome

F-plasmids

facilitates the transfer of genetic material from cell to cell via conjugation

• fertility factors

• type of horizontal transfer

R-factor-Resistance (R) plasmids

resistance transfer factors (RTFs)

• contains genes for conjugation and replication

• antibiotic resistant

tra genes

control the transfer of plasmids from bacterium to bacterium

• important virulence factors

• help with spread and replication

transposable elements (TE)

DNA that can move from one site to another

• jumping genes

Transposable genes do not contain an origin of replication. How do they replicate?

They need to be inserted into another DNA molecule.

TE move via ___.

transposition

All transposons have a specific recombinase necessary for transposition:

transposase

2 types of transposable elements (TE)

1. insertion sequences (IS)

2. transposons

insertion sequence

1. short DNA sequence

2. contain inverted repeats (IR)

Insertion sequences encode for ____.

transposase

• enzyme that allows jumping

transposons

1. longer DNA sequence

2. contain inverted repeats (IR) and transposase

Transposons encode for ____.

additional DNA

types of transposition

1. conservative

   • Transposon is excised.

2. replicative

   • Transposon replicates.

vertical gene transfer

genetic transfer from parents to offspring

• mitosis/meiosis

horizontal/lateral gene transfer

genetic transfer from one organism into another organism that is not its offspring

• can confer antibiotic resistance, virulence, capsule production

transformation

when free DNA released from one cell is taken up by another

transduction

when DNA transfer is mediated by a virus (bacteriophage)

conjugation

when DNA transfer requires cell to cell contact

• has conjugative plasmid in donor cell

homologous recombination

exchange of genes between two DNA molecules resulting in new combinations of genes on a chromosome

endonucleases

cuts/nicks the DNA

RecA protein

catalyzes the joining of the two strands in homologous recombination

heteroduplex

a DNA double helix comprised of single strands from two different DNA molecules

horizontal gene transfer (HGT) transformation

when DNA is taken up from the environment and incorporated into the genome of the recipient

• seen in Griffith Experiment 1927

steps of transformation

1. Dead bacteria release their fragmented genome.

2. Competent bacteria pick up fragments in the environment.

   • Fragments must be short to be picked up by the host cell.

   • Binding DNA

3. Direct uptake of naked dsDNA fragments

   • converted to ssDNA before recombining with the host’s genome

   • Uptake of single-stranded DNA

4. RecA-mediated homologous recombination

Recombination requires:

1. bacterial recombination gene

   • recA, B and C

2. homology between the DNAs

   • donor and recipient must be closely related

competence

the bacterial cell’s ability to absorb naked DNA into the cell

induced competence

when species that are not naturally transformable can be artificially induced

Experiment by Frederick Griffith in 1928

1. Pathogenic strains are killed and release nuclear acid.

2. DNA is taken up by live non-pathogenic strains.

3. DNA converted the non-pathogenic strains into pathogenic strains.

transduction

when DNA is transferred from one bacterial cell to another by a bacteriophage

virulent phages

phages that replicate only via the lytic cycle

temperate phages

phages that replicate using both lytic and lysogenic cycles