Download Prokaryotic Gene Expression

The Genetics of
Viruses and Prokaryotes
Probing the Nature of Genes
• Prokaryotes and viruses have advantages for the study
of genetics:
– They have small genomes.
– They quickly produce large numbers of individuals.
– They are usually haploid, making genetic analyses
easier.
Probing the Nature of Genes
• The ease of use of bacteria and viruses in genetic
research has propelled the science of genetics and
molecular biology during the last 50 years.
• Prokaryotes continue to play a central role as tools for
biotechnology and for research on eukaryotes.
• Prokaryotes play important ecological roles, including
the cycling of elements.
• Many prokaryotes and viruses are pathogens.
Viruses
• TMV - tobacco mosaic virus
– 1st virus to be discovered in the 1890s
• Direct observation of viruses requires electron
microscopes
• The simplest infective agents are viroids, which are
made up only of genetic material.
Viruses
• Viruses are acellular
• Composed of a nucleic acid and a few proteins
– DNA or RNA
– Coat proteins
– Viral enzymes (e.g. reverse transcriptase)
• Do not carry out metabolism
– obligate intracellular parasites
• Reproduce only in living cells
– use host cell’s transcription/translation machinery
– often integrate into host cell’s chromosome(s)
• Progeny released from host cell
– often destroy the host cell in the process
Virions Come in Various Shapes
Adenovirus
TMV
phage
lambda
Influenza A
Virus Nomenclature
• Viruses are categorized by four criteria:
– DNA or RNA genome
– Single-stranded or double-stranded nucleic acid
– Shape of the virion
– Presence or absence of lipid capsule around capsid
Viruses of Prokaryotes
• Bacteriophage - viruses of bacteria
• Bacteriophage recognize hosts via specific interaction
of viral capsid proteins and proteins on host cell.
• Virions are equipped with tail assemblies that inject the
phage’s DNA into the host cell.
Bacteriophage
• Reproduction of a phage involves
– Replication of phage DNA
– Expression of phage genes needed for capsid
• Two types of reproductive cycles
– lytic cycle
• Immediate reproduction and lysis of host cell
– lysogenic cycle
• Integration into host chromosome with reproduction
and lysis occurring later
• Some phage are only lytic other are both (temperate)
• Most well studied is phage Lambda ()
The Lytic and Lysogenic Cycles of Bacteriophage
Plant & Animal Viruses
• Plant viruses are usually only capsid + nucleic acid
• Majority of plant viruses have an RNA genome
• Many animal viruses have a lipid membrane derived from the
host cell’s - envelope
• Some animal viruses have DNA, and some have RNA
• Most viruses are species specific, but many can gain (jump) host
species
– Influenza
– HIV
• Arboviruses infect both insects and vertebrates
Figure 13.4 The Reproductive Cycle of the Influenza Virus (Part 1)
Figure 13.4 The Reproductive Cycle of the Influenza Virus (Part 2)
cDNA
Figure 13.5 The Reproductive Cycle of HIV (Part 1)
Figure 13.5 The Reproductive Cycle of HIV (Part 2)
Viruses can lead to unusual transmission of traits
• Normal genetic transmission
– Vertical transmission
• transfer of genes (traits) from parent to offspring
• Viral mediated gene transfer
– Horizontal transmission
• spread of genes to unrelated individuals
• Horizontal transmission inferred from presence of
transposable elements
– DNA sequences which can move themselves into/out of &
between genomes
– Perhaps incorporated into viruses or perhaps originated from
viruses
Prokaryotes
• Bacteria and archaea
• Single, circular chromosome
– E. coli – 4.65Mbp
• Plasmids
– Extrachromosomal, ds DNA circles
• 1-10Kbp
• Replicated independently of chromosomal DNA
• Contain genes that encode resistance to
antibiotics, metabolic pathways, or conjugation
• Clonal expansion
– Binary fission (prokaryotic cell division)
– Formation of visible colonies on solid media
Figure 13.6 Growing Bacteria in the Laboratory
Prokaryotes: Reproduction and Recombination
• Transformation
– Uptake of DNA in their environment (extracellular
DNA) and incorporation into genome
– Frederick Griffith
– Used by Avery to show DNA was genetic material
• Conjugation
– Prokaryotic “sexual” reproduction
– Physical contact between bacteria and transfer of
plasmids or portions of genome
Figure 13.10 Transformation
Figure 13.7 Lederberg and Tatum’s Experiment - Conjugation
Figure 13.11 Gene Transfer by Plasmids
Figure 13.9 Recombination Following Conjugation
Prokaryotes: Reproduction and Recombination
• Transduction
– viruses carry genes from one cell to another (horizontal
transfer)
– Excision of a prophage to enter a lytic cycle sometime
allows host DNA to be incorporated into the
bacteriophage genome
– Cells infected by such phage get a segment of another
bacterium’s DNA
– This bacterial DNA recombines with the chromosomal
DNA of the host and alters its genetic composition.
Figure 13.10 Transformation and Transduction
Prokaryotes: Reproduction and Recombination
• Transposable elements
– “jumping genes”
– transposons
– segments of DNA that
can move within the
genome
– often contain gene
encoding the enzyme
transposase
Gods
Jacques Monod – Paris 1961
François Jacob & André Lwoff – 1953 CSH Symposium
Regulation of Gene Expression in Prokaryotes
• Metabolic carbohydrate C-sources
– Glucose – feeds directly into glycolysis
– Lactose, Arabinose, Galactose – Feed indirectly into glycolysis
• E. coli only uses secondary sugars once glucose is depleted
• Jacques Monod demonstrated that proteins were induced upon switching C source
– Lactose metabolism - hydrolysis of lactose disaccharide into galactose and
glucose monosaccharides
– The enzyme used is -galactosidase
– Two other enzymes are also involved in lactose metabolism
• A permease to transport lactose into the cell
• An acetylase to modify lactose (unknown biochemical relevance)
• Lactose induces the expression of -galactosidase, permease and acetylase
-Galactosidase Induction by Lactose
Lag
Regulation of Gene Expression in Prokaryotes
• Prokaryotes conserve resources by making proteins only when
needed.
– Lactose metabolic enzymes not made when lactose not present
• Two main ways to regulate metabolic pathways
– Allosteric regulation
• Shape / activity of enzyme
• Protein already present when induction occurs
– Regulation of protein synthesis
• Transcription and /or translation
• Protein made/destroyed when induction occurs
Figure 13.14 Two Ways to Regulate a Metabolic Pathway
Prokaryotic Gene Structure
DNA
Shine-Delgarno
Shine-Delgarno
Stop Codon
Stop Codon
box
+1
box
TAA, TAG, TGA
ATG
ATG
TAA, TAG, TGA
Cis-Regulatory
Coding Sequence= ORF
Coding Sequence= ORF
Elements
USE/Promoter
/Operator
5’ UTR = Leader
sequence
Protein A
Cistron 1
Regulatory Sequences
Spacer = 5’UTR
of 2nd cistron
Protein B
Cistron 2
Structural or Coding Sequences
Regulatory and Coding Sequence Unit = Operon
Terminator
sequence
Prokaryotic Gene Structure I
DNA
Shine-Delgarno
Stop Codon
Shine-Delgarno
Stop Codon
box
TAA,
TAG, TGA
+1
box
ATG
ATG
TAA, TAG, TGA
Cis-Regulatory
Coding Sequence= ORF
Coding Sequence= ORF
Elements
USE/Promoter
/Operator
Protein A
5’ UTR = Leader
sequence
Cistron 1
Terminator
sequence
Cistron 2
5’ UTR = Leader
sequence
Polycistronic
mRNA
Protein B
Spacer = 5’UTR
of 2nd cistron
Spacer
ORF Protein A
+1
AUG
Shine-Delgarno
box
AUG
Stop Codon
Shine-Delgarno
UAA, UAG, UGA
box
ORF Protein B
Stop Codon
UAA, UAG, UGA
The Lac Operon
Regulatory sequences
Transcription & translation of an operon
Transcriptional Regulatory Sequences of Prokaryotes
• Promoter –
– DNA sequences to which RNA polymerase physically
binds
– Two 6 bp elements – -10 box & -35 box
TGTACA
TATAAT
Transcriptional Regulatory Sequences of Prokaryotes
• Operator
– DNA sequence to which a repressor binds
• When repressor is bound, DNA can not be transcribed
• When repressor is not bound transcription proceeds
Repressor
Repressor Proteins: Allosteric Proteins
• The Lac repressor protein has two binding domains
– DNA binding domain
– The inducer (ligand) binding domain
• The lac repressor is a homotetramer protein
– Each monomer provides ½ of a lactose binding site
and ½ of a DNA binding site
– The tetramer binds to DNA in two places or binds to
two lactose molecules
• The repressor can not bind to DNA and lactose
simultaneously
– Binding to lactose alters the shape of the repressor
tetramer causing it to release DNA.
Lac Repressor Tetramer
Lac Repressor-DNA Complex
Lac repressor protein (violet)
forms a tetramer which binds to
two operator sites (red) located
93 bp apart in the DNA causing
a loop to form in the DNA. As a
result expression of the lac
operon is turned off. This model
also shows the CAP (CRP)
protein (dark blue) binding to
the CAP site in the promoter
(dark blue DNA). The -10 and
-35 sequences of the promoter
are indicated in green.
Figure 13.17 The lac Operon: An Inducible System (Part 1)
• Lactose absent – Transcription repressed
– No lacZ, lacY, lacA produced
RNA poly
Figure 13.17 The lac Operon: An Inducible System (Part 2)
Summary of lac operon Transcriptional Control
• When no lactose (inducer) is present, lac operon is off.
• The LacI gene produces the repressor protein
• The repressor prevents transcription of the operon
• The operator is the DNA sequence to which the
repressor binds (binding site)
• The promoter is the DNA sequence to which the RNA
polymerase binds (-10 & -35 boxes)
• Adding inducer (lactose) allows the operon to be
transcribed.
Regulation of Gene Expression in Prokaryotes
• If synthesis of an enzyme can be turned off, it is said to
be repressible.
• The trp operon in E. coli is repressible.
• In the absence of tryptophan, RNA polymerase
transcribes the trp operon, leading to production of
enzymes that synthesize tryptophan.
• When tryptophan is present, it binds to a repressor,
which becomes active.
• The repressor binds to the operator of the trp operon,
blocking production of enzymes for tryptophan
synthesis.
Figure 13.18 The trp Operon: A Repressible System (Part 1)
trp repressor gene
aporepressor
Figure 13.18 The trp Operon: A Repressible System (Part 2)
tryptophan
Inducible or Repressible?
• inducible systems
– Require presence of substrate to activate expression of
operon
– Usually for operons encoding proteins in catabolic
pathways
• repressible systems
– The presence of the substrate inactivates expression of the
operon
– Usually for operons encoding proteins in anabolic
pathways
Control of Transcription in Viruses
• Viruses also have gene regulation mechanisms.
• Bacteriophage  is a temperate phage, meaning that it
can undergo either a lytic or a lysogenic cycle.
• When host bacteria are growing in rich medium, the
prophage remains lysogenic; when the host is less
healthy, the prophage becomes lytic.
• A “genetic switch” determines the prophage behavior.
Lysis vs Lysogeny
• Control of phage gene expression determines life
cycle route
• Study of lytic induction led to the early understanding
of transcriptional regulation
• Phage genome contains a
variety of promoters that
attract host RNA polymerase
to
differing degrees
• Viral control proteins specify
which promoters are used
Map of Phage 
Phage Molecular Biology: Gene Regulation
• Three stages of phage “development”
– Immediate early
• viral genes adjacent to the promoters are transcribed.
– Delayed early
• Proteins of early genes compete to activate/inhibit transcription
of late genes
– Late stages
• Lysis – lytic control proteins win in DE stage – activate lytic
proteins in late stage
• Lysogeny – lysogenic control proteins win in DE stage –
activate prophage formation
Lytic Pathway Chosen
Coat proteins
& lytic enzymes
Transcription factors
DNA
replication
factors
 Phage Gene Expression & Development
Immediate Early
gene expression
Delayed Early
gene expression
Late gene
expression
Figure 13.20 Control of Phage  Lysis and Lysogeny
DNA Sequences Regulating Lytis v Lysogeny
Control of Transcription in Viruses
• cI and cro, compete for two operator/promoter sites on phage DNA.
• The two regulatory proteins have opposite effects when bound to the
operators.
– cI represses the lytic promoter and activates the lysogenic promoter
– cro activates the lytic promoter and represses the lysogenic promoter.
• The relative concentrations of cI and cro determine the outcome.