Exam #2 Flashcards
Genome
genome: complete set of genetic information
chromosome plus plasmids
all cells: DNA
(viruses may have RNA)
functional unit is gene
encodes gene product, usually a protein
study of nucleotide sequence is genomics
bacterial genome
bacterial chromosome: circular molecule of DNA
- a self-replicating genetic element
extra-chromosomal genetic elements: plasmids
nonessential replicons
resistance to antimicrobial agents or production of virulence factors
Central dogma
The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
DNA
Incredible diversity of life determined by information within DNA
composed of four nucleotides:
adenine (A), thymine (T), cytosine (C) , guanine (G)
DNA can code for enormous amount of information
3 nucleotides encode specific amino acid
amino acids make up protein
sequence determines structure
DNA, RNA initially synthesized as ribonucleotides
purines: atoms added to ribose 5-phosphate to form ring
pyrimidines: ring made, then attached to ribose 5-phosphate
can be converted to other nucleobases of same type
Purines: Adenine Guanine double ring
Pyrimidine: Thymine Cytosine Uracil single ring
Base pairing
nucleotides joined between 5′PO4 and 3′OH with ester link
forms sugar-phosphate backbone
single DNA strand will have a 5′ and 3′ end
strands are complementary and antiparallel
held together by hydrogen bonds between nucleobases
base-pairing:
cytosine (C) to guanine (G) (three hydrogen bonds)
adenine (A) to thymine (T) (two hydrogen bonds)
separating strands called melting or denaturing
characteristics of RNA
RNA (ribonucleic acid)
ribose instead of deoxyribose
uracil in place of thymine
usually shorter single strand
synthesized from DNA template strand
RNA molecule is transcript
base-pairing rules apply except uracil pairs with adenine
transcript quickly separates from DNA
characteristics of RNA
RNA (ribonucleic acid)
three types required for gene expression
messenger RNA (mRNA)
ribosomal RNA (rRNA)
transfer RNA (tRNA)
DNA replication
DNA replication usually bidirectional
creates two replication forks
ultimately meet at terminating site when process complete
replication is semiconservative
In the two new molecules generated, each has one new strand and one original strand
replication begins at origin of replication
proteins recognize and bind to site
melt double-stranded DNA
oriC region characteristics
Replication is initiated through cooperative binding of the initiator protein, DnaA, to multiple DnaA-recognition sites within the oriC region.
SeqA strictly prevents the initiation of new rounds of replication via a mechanism called “sequestration.” SeqA inhibits replication initiation by blocking DnaA from binding.
Fis negatively influences replication initiation by regulating the occupation of DnaA.
IHF binding leads to bending of the DNA.
This triggers separation of the DNA strands at the AT-rich DNA unwinding element (DUE), providing an entry site for helicase and later on the other enzymes (e.g., primase and DNA Pol III) that are responsible for DNA synthesis.
In circular DNA, bidirectional replication from an origin leads to the formation of replication intermediates resembling the Greek letter theta.
Primase
primases synthesize short stretches of complementary RNA called primers
At ORI site, two leading strands primed, one in each direction
Primers are required for DNA synthesis because no known DNA polymerase is able to initiate polynucleotide synthesis. DNA polymerases are specialized for elongating polynucleotide chains from their available 3′-hydroxyl termini. In contrast, RNA polymerases can elongate and initiate polynucleotides.
Primer: initiation of DNA synthesis
process of DNA replication
DNA polymerases synthesize in 5′ to 3′ direction
hydrolysis of high-energy phosphate bond powers
DNA polymerase can only add nucleotides, not initiate
require primers at origin of replication
helicases “unzip” DNA strands
reveals template sequences
leading strand synthesized continuously
lagging strand synthesized discontinuously
DNA polymerases can only add nucleotides to 3′ end
production of Okazaki fragments
different DNA polymerase replaces primers
DNA ligase forms covalent bond between adjacent nucleotides
bacterial chromosome
Origin and terminus of replication divide genome into oppositely replicated halves
1 – replicated clockwise
has presented strand of E. coli as
leading strand
2 – complementary strand is leading one.
Transcription
RNA polymerase synthesizes single-stranded RNA
uses DNA template
synthesizes in 5′ to 3′ direction
can initiate without primer
binds to promoter
found upstream of genes
stops at terminator
transcription ends
transcription
RNA polymerase uses DNA template to synthesize single-stranded RNA transcript in 5’ to 3’ direction
transcription
RNA sequence is complementary, antiparallel to DNA template strand
DNA template is minus (–) strand
complement is plus (+) strand
RNA has same sequence as (+) DNA strand except uracil instead of thymine
mRNA transcripts are MONOCISTRONIC (code for one gene)
OR
POLYCISTRONIC (code for multiple genes)…
Sigma (σ) factor recognizes promoter
subunit loosely attached to RNA polymerase
various types of sigma factors recognize different promoters
synthesis controls transcription of sets of genes
eukaryotic cells, archaea use transcription factors
Initiation of transcription begins with promoter binding by RNA polymerase holoenzyme.
holoenzyme = RNA polymerase core + sigma factor
Promoters
promoter orients the direction of transcription in one of two directions.
By doing so, it also determines which strand is the template for the transcript.
found upstream of genes
once RNA polymerase has moved past, another RNA polymerase can bind
allows rapid and repeated transcription of single gene
Operon
remember: bacteria may make polycistronic (polygenic) mRNAs
An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter.
Why is knowing the orientation of a promoter critical when determining the amino acid sequence of an encoded protein?
The promoter orients the RNA polymerase in one of two directions.
By doing so, it also determines which strand is the template for the transcript.
Translation
genetic code: three nucleotides = codon
redundancy: code is degenerate
three reading frames possible
depends on start of coding region
correct reading frame is critical
incorrect will yield different, likely nonfunctional protein
translation in prokaryotes begins before transcription is complete
Ribosomes and translation
ribosomes serve as translation “machines”
prokaryotic comprised of 30S and 50S subunits
made from protein and ribosomal RNA (rRNA)
locate punctuation sequences on mRNA molecule
begins at start site, moves along in 5′ to 3′
maintain correct reading frame
aligns and forms peptide bond between amino acids
tRNA
transfer RNA (tRNAs) deliver correct amino acid
-has specific anticodon sequence
-base-pairs with correct codon
-carries appropriate amino acid
after delivering, tRNA can be recycled
enzyme in cytoplasm recognizes tRNA and attaches appropriate amino acid
translation initiation
part of ribosome binds to mRNA sequence
termed *ribosome-binding site
first AUG after that site serves as start codon
complete ribosome assembles at start codon
initiating tRNA brings altered form of methionine
occupies P-site
(peptidyl-site)
Ribosome has two sites to which tRNAs can bind
P-site occupied by tRNA carrying methionine
another tRNA recognizes codon in empty A-site
occupies A-site, brings correct amino acid
A-site and P-site now occupied by correct tRNAs
enzyme creates peptide bond between their amino acids
amino acid from tRNA in P-site added to amino acid carried by tRNA in A-site
Elongation (translation)
elongation of polypeptide chain
ribosome advances along mRNA in 5′ to 3′ direction
initiating tRNA exits through E-site
remaining tRNA carrying both amino acids occupies P-site
A-site transiently empty
a tRNA that recognizes codon in A-site quickly attaches
peptide bond formed between amino acids
ribosome advances one codon on mRNA
tRNA exits E-site, new tRNA occupies A-site
process repeats
once ribosome clears initiating sequences, another ribosome can bind: polyribosome, or polysome
Termination (translation)
termination
elongation continues until ribosome reaches stop codon
not recognized by tRNA
enzymes free polypeptide
break covalent bond joining to tRNA
freed ribosome falls off mRNA
disassociates into component subunits (30S and 50S)
subunits can be reused to initiate translation at other sites
Amino acid synthesis much slower than DNA synthesis
signal transduction
The regulation of gene expression is influenced by external and internal molecular cues and/or signals.
Sensing and responding to environmental fluctuations
Single-celled organisms with short doubling times must respond extremely rapidly to their environment.
Bacteria are exposed to changing conditions and must be able to adapt to stresses such as nutrient limitation, temperature shifts, varying osmolarity, and transition from exponential growth to stationary phase
Adaptation involves changes in gene expression
signal transduction
transmits information from outside cell to inside
allows cells to monitor and react
Regulation of protein expression in bacteria mostly occurs at the level of transcription of genes, carried out by RNA polymerase (RNAP) by binding to specific regions on the chromosome (promoters).
Quorum sensing
microorganisms constantly face changing environment
must adapt quickly to survive
quorum sensing
some organisms can “sense” density of their population
allows cells to activate genes useful with critical mass
for example, biofilm formation, pathogens′ infective process
quorum sensing is a method of cell-to-cell communication
affects gene expression
and physiological behavior of microbial communities.
Production of signal molecules continuous for each cell, BUT…responses only initiated when signal reaches threshold concentration.
quorum of bacteria required to produce signal concentrations above threshold; as population increases, concentration of signal increases because more signal producers present
some can detect, interfere with signaling molecules of other species
can “eavesdrop” and obstruct “conversations”
examples:
bioluminesence
biofilm formation
pathogens′ infective process
quorum sensing - Pseudomonas aeruginosa
Gram-negative
capable of surviving in wide range of environments.
opportunistic pathogen
commonly associated with nosocomial infections
burn wound infections
leading cause of death in severe respiratory infections (cystic fibrosis)
quorum sensing has key role in pathogenesis of P. aeruginosa
regulates production of extracellular virulence factors
regulates expression of antibiotic efflux pumps
promotes biofilm maturation
Infections with P. aeruginosa difficult to eradicate, due to antibiotic resistance and growth in biofilms.
biofilm formation
switch from single-cell, planktonic lifestyle to multicellular, sessile biofilm involves changes in gene expression to produce:
adhesins
extracellular polysaccharide-containing matrix
flagella formation
quorum sensing in Gram positive cells
Quorum sensing in Gram positive cells – small peptides (autoinducing peptides)
Peptide signals are not diffusible across the membrane
two-component regulatory systems
membrane-spanning sensor
modifies internal region in response to
specific environmental variations
phosphorylates amino acid
histidine kinase sensor protein
response regulator
phosphate group transferred from sensor
regulator turns genes on or off in response
and a transcriptional regulator known as response regulator
Natural selection
natural selection can play role in gene expression
expression of some genes changes randomly in cells
enhances survival of at least part of population
Antigenic variation
antigenic variation - alteration of characteristics of surface proteins - allows pathogens to stay ahead of host defenses
Neisseria gonorrhoeae - many genes for pilin (protein subunit of pili)
only expresses gene in expression locus
randomly moves genes in and out of expression locus
immune system responds to dominant pilin type
bacteria that have “switched” type survive
Phase variation
phase variation involves switching genes on and off
allows E. coli to attach via pili, detach by turning off
regulation of gene expression
-except for “house-keeping” genes, most genes expressed or repressed depending on specific conditions under which cells grow.
-competition for scarce resources (nutrients) makes bacterial cells efficient
Regulating gene expression is one way to save energy.
-gene activity controlled by promoter and regulatory elements that determine whether RNAP will transcribe gene
bacterial gene regulation
Genes can be routinely expressed or regulated
a set of regulated genes transcribed as single mRNA along with its control sequences is termed operon
-lac operon for lactose metabolism
separate operons controlled by single regulatory mechanism constitute regulon
global control is simultaneous regulation of numerous genes
Type of regulation
enzymes can be grouped by type of regulation
constitutive enzymes synthesized constantly
typically indispensable roles in central metabolism (enzymes of glycolysis)
inducible enzymes not routinely produced
Synthesize only when needed (β-galactosidase turned on only when lactose present)
avoid waste of resources
repressible enzymes
produced routinely
turned off when not required
(anabolic pathways such
as amino acid synthesis)
What is the difference between inducible gene expression and repressible gene expression?
Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression.
Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.
mechanisms to control transcription
must be readily reversible, allow cells to control relative number of transcripts produced
two most common are alternative sigma factors and DNA-binding proteins
Sigma factors
alternative sigma factors
standard sigma factor (σ70 - sigma factor with molecular weight of 70 - “housekeeping” sigma factor or primary sigma factor, transcribes most genes in growing cells. Every cell has a “housekeeping” sigma factor that keeps essential genes and pathways operating)
is loose component of RNA polymerase that recognizes specific promoters for genes expressed during routine growth conditions
Alternate sigma factors recognize promoters of different architectures – different regulons of different genes; with alternative sigma factors RNAP redirected to new sets of genes
Sigma 70 - housekeeping sigma factor - association with RNAP favored because high intracellular level and higher affinity to core RNAP
Most housekeeping genes expressed during exponential growth transcribed by holoenzyme containing σ70 and RNA polymerase
Alternative σ factors provide a line of response to fluctuating changes in their environment such as heat shock, variation in pH, and osmolarity, nutrient deprivation by effectively reprograming the transcription of sets of specific genes
-sporulation in Bacillus subtilis controlled by multiple different alternative sigma factors
DNA binding proteins: repressors
DNA-binding proteins can act as repressors or activators
repressor blocks transcription (negative regulation)
binds to operator, stops RNA polymerase
repressors are allosteric: have binding site that alters ability to bind to DNA
two general mechanisms
induction: repressor binds, blocks transcription
inducer binds to repressor, repressor unable to bind
Inducible - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule induces gene expression.
repression: repressor unable to bind to DNA
corepressor attaches to repressor, complex now binds to DNA and blocks transcription
Repressible - A repressible system is on except in the presence of some molecule (called a co-repressor) that suppresses gene expression. The molecule is said to repress expression.
DNA binding proteins: activators
activator facilitates transcription (positive regulation)
ineffective promoter preceded by activator-binding site
binding of activator enhances ability of RNA to initiate transcription at promoter
inducer binding to activator allows binding to DNA///
Transcription control
DNA-binding proteins can act as repressors or activators
repressor blocks transcription (negative regulation)
binds to operator, stops RNA polymerase
repressors are allosteric: have binding site that alters ability to bind to DNA
two general mechanisms
induction: repressor binds, blocks transcription
inducer binds to repressor, repressor unable to bind
repression: repressor unable to bind to DNA
corepressor attaches to repressor, complex now binds to DNA and blocks transcription
lac operon –
inducible gene expression
lactose and the lac operon
no lactose: repressor prevents transcription
lactose present: some converted to inducer allolactose
binds to repressor
repressor releases
operator
RNA polymerase
transcribes operon
only occurs when
glucose unavailable
mechanisms to control transcription – lac repressor
No lactose: lac operon proteins not made, because not needed.
If lactose, inducer binds to lac repressor, allows transcription of lac operon genes.
When lactose depleted, lac repressor loses inducer, and blocks production of proteins, since they are no longer needed.
Glucose and the lac operon
glucose and the lac operon
carbon catabolite repression (CCR) prevents expression of lac operon in presence of glucose
prioritize carbon/energy sources; yields diauxic growth
glucose transport system senses glucose
catabolite activator protein (CAP) required for transcription
functional only when bound by inducer cAMP
cAMP made when glucose low
Inducer exclusion: lactose transporter
blocked during glucose transport
cyclic adenine monophosphate (cAMP) involved in positive regulation of lac operon
transcription factor CAP (catabolite gene activator protein) forms complex with cAMP (inducer) and is activated to bind to DNA
cAMP level varies, depending on growth medium
cAMP low when glucose is carbon source:
glucose causes inhibition of adenylate cyclase, the enzyme that produces cAMP
Structural basis for cAMP-mediated allosteric control of the catabolite activator protein (CAP)
low glucose concentration
cAMP accumulates
binds allosteric site on CAP
CAP assumes active shape
binds upstream of lac promoter
makes it easier for RNA polymerase to bind promoter and start transcription of lac operon
increases rate of lac operon transcription
high glucose concentration
cAMP concentration decreases
CAP disengages from lac operon
high glucose concentration
cAMP concentration decreases
CAP disengaged from lac operon
low glucose concentration
cAMP accumulates
binds allosteric site on CAP
CAP assumes active shape
binds site upstream of lac promoter
makes it easier for RNA polymerase to bind adjacent promoter and start transcription of the lac operon
increases rate of lac operon transcription.
lac operon regulation by CAP (activator) and lac repressor
No lactose, no glucose
No glucose, lactose present
Glucose present, no lactose
Lactose present, glucose present
(Know the diagram)
Beta galactosidase
Beta galactosidase hydrolyzes lactose to produce glucose and galactose
(lac Z)
Lactose is both inducer for beta-galactosidase (lac Z) expression, and, when expressed, is its substrate
IPTG
experimental inducer - not hydrolyzed by beta-galactosidase
IPTG able to induce the operon, but it cannot be hydrolyzed by beta-galactosidase.
(IPTG can bind repressor to change its conformation so it will not bind at operator.)
induction of gene and performance of gene’s function can be separated.
genomics
analyzing a prokaryotic DNA sequence
(+) strand used to represent sequence of RNA transcript
ATG in (+) DNA indicates possible start codon
computers search for open reading frames (ORFs)
stretches of nucleotides generally longer than 300 bp
begin with start codon, end with stop codon
ORF potentially encodes protein
other characteristics (upstream sequence serving as ribosome-binding site) also suggest ORF encodes protein
can be compared with published sequences
presumed function can be assigned
Metagenomics
analysis of total microbial genomes in environment
can study all microorganisms and viruses in community
not limited to just those that grow in culture
can track changes in composition of microbiota of individual over time (healthy, diseased)
compare microbiota at different body sites
also between different individuals
study microbial life in oceans, soils
new understanding of extent of biodiversity
may lead to discoveries of useful compounds (for example, antibiotics)
Tremendous amount of data presents challenges
mutation as a mechanism of genetic change
spontaneous mutations: base substitutions, deletion or addition of nucleotides, transposons
induced mutations: chemical mutagens, transposition, radiation
repair of damaged DNA
proofreading (DNA polymerase), mismatch repair, modified bases, thymine dimers, SOS repair
mutant selection: direct and indirect selection, mutant screening
Horizontal gene transfer
transformation competence, Griffith’s experiment
transduction
conjugation
mobile gene pool transposons, plasmids, genomic islands, phage DNA
genetic change in bacteria
by mutation or by horizontal gene transfer
Why do genetic changes, like mutations, have a more dramatic impact on bacteria relative to more complex organisms like humans?
A genetic change alters an organism’s genotype.
gene mutations -
spontaneous and induced
This can have a profound impact on bacteria because they are haploid.
Because of this, a change in genotype can easily alter the observable characteristics of an organism, its phenotype
Mutations can change organism’s phenotype
deletion of gene for tryptophan biosynthesis yields mutant that only grows if tryptophan supplied
growth factor required; mutant termed auxotroph
geneticists compare mutants to wild type
typical phenotype of strains isolated from nature
wild-type E. coli strain is prototroph
strains designated by three-letter abbreviations
for example, Trp– cannot make tryptophan
streptomycin resistance designated StrR
Autotrophs and prototrophs
An auxotroph requires an organic growth factor in order to grow.
A prototroph has no such requirement
stochastic
randomly determined; having a random probability distribution or pattern that may be analyzed statistically but may not be predicted precisely.
Mutations
mutations passed to progeny
occasionally change back to original state: reversion
large populations contain mutants (example: cells in colony)
environment selects cells that grow under its conditions
-antibiotics select for resistant bacteria if present
-environment does not cause mutations*
-single mutation rare; two even rarer
-physicians may use two antibiotics to reduce resistance
chance is product of mutation rate for each
Replica plating
Replica plating: reproduce colonies from an original plate to new plates by “stamping” the original plate with velvet then stamping empty plates with the same velvet. Bacteria from each colony are picked up then deposited on new plates by the velvet.
Did the colonies on the new plate evolve antibiotic resistance?
Result:
Penicillin-resistant bacteria were in population before they encountered penicillin, they did not evolve resistance in response to exposure to the antibiotic.
…the environment does not cause mutations…spontaneous mutations caused by normal processes occur randomly at infrequent characteristic rates….
kinds of spontaneous mutations
base substitution
addition or deletion
transposable elements
Base substitution
base substitution most common
incorrect nucleotide incorporated
during DNA synthesis
point mutation is change
of a single base pair
base substitution leads to three possible outcomes
silent mutation: wild-type amino acid
missense mutation: different amino acid
resulting protein may only partially function
termed leaky
nonsense mutation:
specifies stop codon
yields shorter protein
base substitutions may affect translation
genetic code: three nucleotides = codon
redundancy: code is degenerate
Base substitution
base substitutions
a mutation that inactivates gene is termed a null or knockout mutation
“silent mutation” sometimes used to indicate mutation that does not alter protein function
base substitutions more common in aerobic environments
reactive oxygen species (ROS) produced from O2
can oxidize nucleobase guanine
DNA polymerase often mispairs with adenine
deletion or addition of nucleotides
impact depends on number of nucleotides
three pairs changes one codon
one amino acid more or less
impact depends on location in protein
one or two pairs yields
frameshift mutation
different set of codons translated
-premature stop codon
shortened, nonfunctional protein
knockout mutation
A frameshift mutation affects all the amino acids inserted after the frameshift occurs
Why is deleting one nucleotide generally more detrimental than deleting three?
Deleting one nucleotide results in frame shift , resulting in a change of all amino acids translated beyond the deletion.
Deleting three nucleotides results in only the deletion of one amino acid.
Transposons
transposons (jumping genes)
can move from one location to another
process is transposition
gene insertionally inactivated
function destroyed
most transposons have transcriptional terminators
blocks expression of downstream genes in operon
induced mutations
induced mutations result from outside influence
agent that induces change is mutagen
geneticists may use mutagens to increase mutation rate
two general types: chemical, radiation
chemical mutagens may cause base substitutions or frameshift mutations
some chemicals modify nucleobases
change base-pairing properties
increase chance of incorrect nucleotide incorporation
nitrous acid (HNO2) converts cytosine to uracil
base-pairs with adenine
instead of guanine
induced mutations – alkylating agents
Alkylating agents add alkyl groups onto nucleobases
nitrosoguanidine adds methyl group to guanine
base-pairs with thymine
induced mutations – base analogs
base analogs resemble nucleobases
have different hydrogen-bonding properties
can be mistakenly incorporated by DNA polymerase
5-bromouracil resembles
thymine, often base-pairs
with guanine
2-amino purine resembles
adenine, often pairs with
cytosine
Intercalating agents cause frameshift mutations
flat molecules that intercalate (insert) between adjacent base pairs in DNA strand
pushes nucleotides apart, produces space
causes errors during replication
if in template strand, base pair added to synthesized strand
if in strand being synthesized, a base pair deleted
-often results in premature stop codon
ethidium bromide is common intercalating agent
carcinogen
Induced mutations- transposition
transposons can be used to generate mutations
transposon inserts into cell’s genome
generally inactivates gene into which it inserts
induced mutations - radiation: two types
- ultraviolet irradiation forms thymine dimers
covalent bonds between adjacent thymine
cannot fit into double helix; distorts molecule
replication and transcription stall at distortion
cell will die if damage not repaired
mutations result from cell’s SOS repair mechanism
X rays cause single and
double-strand breaks in DNA
double-strand breaks
often produce lethal deletions
X rays can alter nucleobases
repair mechanisms
Enormous amount of spontaneous and mutagen-induced damage to DNA
if not repaired, can lead to cell death; cancer in animals
in humans, two genes associated with breast cancer code for DNA repair enzymes; mutations in either result in 80% probability of breast cancer
mutations are rare; alterations in DNA generally repaired before being passed to progeny
several different DNA repair mechanisms
Mechanisms for repair of damaged DNA
proofreading
mismatch repair
light repair (photoreactivation) and dark repair (excision repair)
glycosylases
SOS repair
BRCA 1 and 2 are human DNA repair factors – pathway repairs double stranded breaks in DNA
repair of errors in nucleotide incorporation
During replication, DNA polymerase sometimes incorporates wrong nucleotide
mispairing slightly distorts DNA helix
recognized by enzymes
mutation prevented by repairing before DNA replication
two mechanisms: proofreading, mismatch repair
proofreading by DNA polymerase
verifies accuracy
can back up, excise nucleotide
incorporate correct nucleotide
very efficient but not flawless
What is proofreading
The removal of an incorrect base and the incorporation of the correct base in its place.
In bacteria, DNA polymerases have the ability to proofread, using 3’ → 5’ exonuclease activity. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base.
Mismatch repair
fixes errors missed by DNA polymerase
enzyme cuts sugar-phosphate backbone
another enzyme degrades short region of DNA strand
methylation of DNA indicates template strand
methylation takes time, so newly synthesized strand is unmethylated
DNA polymerase, DNA ligase make repairs
mismatch repair
DNA mismatch repair (MMR) corrects mismatched base pairs mainly caused by DNA replication errors.
The mechanisms and proteins involved in the reactions of MMR are highly conserved from bacteria to humans.
modified nucleobases lead to base substitutions
glycosylase removes
oxidized nucleobase
another enzyme cuts
DNA at this site
DNA polymerase
removes short section;
synthesizes replacement
DNA ligase seals gap
repair of thymine dimers
Several methods to repair damage from UV light
photoreactivation: light repair
enzyme uses energy from light
breaks covalent bonds of thymine dimer
only found in bacteria
excision repair: dark repair
enzyme removes damage
DNA polymerase, DNA ligase repair
SOS repair: repair of thymine dimers
SOS repair: last-ditch repair mechanism
SOS response - inducible DNA repair system
-allows bacteria to survive sudden increases in DNA damage
induced following extensive DNA damage
photo-reactivation, excision repair unable to correct
DNA and RNA polymerases stall at unrepaired sites
several dozen genes in SOS system activated
includes a DNA polymerase that synthesizes even in extensively damaged regions
has no proofreading ability, so errors made
result is SOS mutagenesis
LexA repressor regulates transcription of all SOS genes.
Why would a cell use SOS repair, considering that it introduces mutations?
Without SOS repair, the cell would not be able to multiply.
mutant selection
Mutants rare, difficult to isolate
two main approaches
direct selection: cells inoculated onto medium that supports growth of mutant but not parent
-antibiotic-resistant mutants exposed to antibiotic
Mutant selection – antibiotic resistance – direct selection
Types of mutant selection
Mutants rare, difficult to isolate
two main approaches
direct selection: cells inoculated onto medium that supports growth of mutant but not parent
-antibiotic-resistant mutants exposed to antibiotic
indirect selection: isolates
auxotroph from prototrophic
parent strain
more difficult since parents will grow on any media on which
auxotroph can grow
replica plating
How to isolate lactose mutants? (indirect selection)
take 1 x 109 cells/ml E. coli
treat with chemical mutagen (such as EMS – ethylmethane sulfonate - alkylating agent)
plate on glucose plate
replica plate onto medium with lactose - only carbohydrate source
mutant unable to grow (would be lac- mutant)
Penicillin enrichment
penicillin enrichment of mutants sometimes used
increases proportion of auxotrophs in broth culture
penicillin kills growing cells
prototrophs
auxotrophs survive
penicillinase then added
cells plated on rich medium
Ames test
screening for possible carcinogens
carcinogens cause many cancers; most are mutagens
animal tests expensive, time-consuming
mutagens increase low frequency of spontaneous reversions
Ames test measures effect of chemical on reversion rate of histidine-requiring Salmonella auxotroph
uses direct selection
if mutagenic, reversion rate increases relative to control
rat liver extract added since non-carcinogenic chemicals often converted to carcinogens by animal enzymes
additional tests on mutagenic chemicals to determine if carcinogenic
Microorganisms commonly acquire genes from other cells: horizontal gene transfer
can demonstrate recombinants
with auxotrophs
combine two strains
for example, His–, Trp– with Leu–, Thr–
spontaneous mutants unlikely
colonies that can grow on
glucose-salts medium most likely
acquired genes from other strain
Genes naturally transferred by three mechanisms
transformation: naked DNA uptake by bacteria
transduction: bacterial DNA transfer by viruses
conjugation:
DNA transfer during cell-to-cell contact
horizontal gene transfer as a mechanism of genetic change
DNA replicated only if replicon
has origin of replication
plasmids, chromosomes
DNA fragments added to chromosome via homologous recombination
only if sequence similar to region of recipient’s genome
DNA-mediated transformation
naked DNA not within cell or virus
cells release DNA when lysed
->addition of DNase prevents transformation
demonstration of transformation in S. pneumoniae
only encapsulated cells pathogenic
CAPSULES: Rough strain doesn’t form capsule = rough colonies on plate
Smooth strain forms a capsule
shiny round colonies on plate
demonstration of transformation in Streptococcus pneumoniae
only encapsulated cells pathogenic!
Competence
transformation
recipient cell must be competent
most take up regardless of origin
some accept only from closely related bacteria (DNA sequence)
process tightly regulated
Streptococcus pneumoniae has two-component regulatory system
high concentration bacteria (quorum sensing)
only fraction of population becomes competent
MECHANISM OF DNA UPTAKE during transformation
- Double stranded DNA binds to plasma membrane
- Enzymes cut the DNA
- Broken down to one strand
- Protein binds the DNA and replaces part of the bacterium DNA
dsDNA bound to the cell surface
fragmentation of dsDNA occurs upon binding
ssDNA fragments transported across the membrane via transformation pseudopilus, evolutionarily related to type IV pili
transport possibly driven by proton motive force.
retraction (disassembly) of pseudopilus allows exogenous DNA to cross peptidoglycan
RecA protein
RecA gene family plays central role in homologous recombination during bacterial transformation
The RecA protein is essential for transformation in Bacillus subtilis and Streptococcus pneumoniae . Expression of the RecA gene is induced during development of competence for transformation in these organisms.
RecA protein interacts with entering single-stranded DNA (ssDNA) to form RecA/ssDNA nucleofilaments that scan the chromosome for regions of homology and bring the entering ssDNA to the corresponding region, where strand exchange and homologous recombination occur.
Transduction
bacterial DNA transfer by viruses (bacteriophages)
transfer of bacterial DNA enclosed in a phage head
from one bacterium to another
generalized transduction: any genes of donor cell
specialized transduction: specific genes
transfer of old bacterial host DNA to new bacterial host via bacteriophage
Conjugation
conjugation: DNA transfer between bacterial cells via specialized conjugal pilus
requires contact between donor, recipient cells
pilus - structure used by bacteria during conjugation
transfer of genetic material between a donor and a recipient cell
conjugative plasmids direct their own transfer
replicons
F plasmid (fertility) of E. coli most studied
other plasmids encode resistance to some antibiotics
spread resistance easily via conjugation
conjugation
F plasmid of E. coli
F+ cells have, F– do not
encodes proteins, including
F pilus
sex pilus
brings cells into contact
enzyme cuts plasmid
single strand transferred
complementary strands
synthesized
both cells are now F+
F plasmid also contains an origin of transfer.
chromosomal DNA transfer via conjugation is less common
involves Hfr cells (high frequency of recombination)
F plasmid is integrated into chromosome via homologous recombination
process is reversible
F′ plasmid results when small piece of chromosome is removed with F plasmid DNA
F′ is replicon
Hfr cell produces F pilus
transfer begins with genes on one side of origin of transfer of plasmid (in chromosome)
part of chromosome transferred to recipient cell
chromosome usually breaks before complete transfer (full transfer would take ~100 minutes)
recipient cell remains F– since incomplete F plasmid transferred
Relationship between high cell density and hozizontal gene transfer?
Quorum sensing mediates coordinated shifts in group behavior – conjugation and competence
mobile gene pool
Genomics reveals surprising variation in gene pool of even a single species
perhaps 75% of E. coli genes found in all strains
termed core genome of species
remaining make up mobile gene pool
plasmids, transposons, genomic islands, phage DNA
plasmids found in many bacteria and archaea some eukarya
usually dsDNA with origin of replication - generally nonessential; cells survive loss
Resistance plasmids
resistance to antimicrobial medications, heavy metals (mercury, arsenic)
often two parts
R genes
RTF (resistance transfer factor)
codes for conjugation
often broad host range
normal microbiota can transfer to pathogens
Transposons
transposons provide mechanism for moving DNA
can move into other replicons in same cell
simplest is insertion sequence (IS)
encodes only transposase enzyme, inverted repeats
composite transposons include one or more genes
integrate via
non-homologous recombination
Transposons yielded vancomycin-resistant Staphylococcus aureus strain
patient infected with S. aureus
susceptible to vancomycin
also had vancomycin-resistant
strain of Enterococcus faecalis
-transferred transposon-containing plasmid to S. aureus
bacteria can conjugate with plants
natural genetic engineering
Agrobacterium tumefaciens causes crown gall
different properties, produces opine, plant hormones
piece of tumor-inducing (Ti) plasmid called T-DNA (transferred DNA) is transferred to plant, incorporated into plant chromosome
Genomic islands
genomic islands: large DNA segments in genome
originated in other species
nucleobase composition very different from genome
G-C base pair ratio characteristic for each species
may provide different characteristics:
utilization of energy sources
acid tolerance
development of symbiosis
ability to cause disease
pathogenicity islands
restriction modification systems
resisting phage infection by degrading foreign DNA
restriction enzymes and a modification enzyme
restriction enzyme: recognizes short nucleotide sequence then cuts at that sequence
modification enzyme protects cells own DNA by adding methyl groups to self DNA
restriction enzymes can’t degrade methylated (self) DNA
CRISPR
clustered regularly interspaced short palindromic repeats
Some bacterial genomes include very small pieces of phage DNA.
The bacteria have survived phage infections and retained small segments of that invader’s DNA, incorporated into the bacterial genome.
Segments are used to recognize and destroy that specific invading DNA in the future, providing the cell with a form of adaptive immunity.
Bacterial defenses against phages
three mechanisms confer bacterial resistance to phage infection:
- preventing phage attachment
- restriction/modifiction systems
- CRISPR
Bacteria and their viral predators (bacteriophages) locked in constant battle.
To proliferate in phage-rich environments, bacteria have impressive arsenal of defense mechanisms.
In response, phages have evolved counter-strategies to evade these antiviral systems.
- preventing phage attachment
Recognition of host receptor molecules
A limiting factor for phage invasion of a bacterial cell is the ability of the phage to bind to a receptor on the host bacterial cell surface.
If a bacterium alters or covers a receptor, that cell becomes resistant to any phage that requires the receptor for attachment.
Bacteria can lose or alter the target receptor of phages
Can produce an extracellular matrix of polysaccharides that blocks phage attachment
Can produce competitive inhibitors that bind to the phage attachment site.
phage response to bacterial defenses
- Epithelial cells secrete mucus.
- Phage adhere to mucus through Iq-like domains.
- Adherent phage form
anti-microbial layer. - Mucus-adherent phage
have increased chance of
replicative success - Phage and bacteria are shed with mucus.
- restriction modification systems
resisting phage infection by degrading foreign DNA
restriction enzymes and a modification enzyme
restriction enzyme: recognizes short nucleotide sequence then cuts at that sequence
modification enzyme protects cells own DNA by adding methyl groups to self DNA
restriction enzymes can’t degrade methylated (self) DNA
- CRISPR
clustered regularly interspaced short palindromic repeats
Some bacterial genomes include very small pieces of phage DNA.
The bacteria have survived phage infections and retained small segments of that invader’s DNA, incorporated into the bacterial genome.
Segments are used to recognize and destroy that specific invading DNA in the future, providing the cell with a form of adaptive immunity.
CRISPR - 3 steps:
invader DNA incorporation
CRISPR array transcribed and processed by Cas proteins
CRISPR interference: the invader’s DNA is recognized by complementarity to the crRNA and is neutralized
Summary:
Viruses are obligate intracellular parasites that collectively can infect all forms of life.
Bacteriophages are viruses that infect bacteria.
Q: Describe three mechanisms that confer bacterial resistance to phage infection.
A: 1. Preventing phage attachment
2. Restriction/Modification systems
3. CRISPR
Q: How do modification enzymes protect host cell DNA from restriction enzymes?
A: By methylating certain nucleobases which then are not recognized by the restriction enzyme.
Q: How does a bacterial cell acquire a historical record of phage infections?
A: By incorporating a piece of the entering phage genome into its own genome.
applications of genetic engineering
Many uses for genetically engineered bacteria:
protein production
DNA production
research tools
relies on DNA cloning
Molecular cloning
…a set of techniques used to insert recombinant DNA from a prokaryotic (or eukaryotic) source into a replicating vehicle such as a plasmid or a viral vector.
Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene.
biotechnology
Use of microbiological and biochemical techniques to solve practical problems
recombinant DNA techniques
possible to genetically alter organisms
can isolate genes from one, transfer to another
genetic engineering
numerous uses: agriculture, medicine, law enforcement
Restriction enzymes
-recognize 4 to 6 base-pair nucleotide sequence and cut each strand of DNA
-typically palindrome
-generates restriction fragments
-cohesive ends (sticky ends) are complementary, can anneal
-allows creation of recombinant DNA molecules
Mix the two ends in the presence of ATP and an enzyme, T4 DNA ligase. *T4 bacteriophage
During ligation reaction, hydrogen bonds form between overhangs on the fragments, then ligase repairs phosphate backbone (phosphodiester bond)
Plasmids and vectors
plasmids - circular pieces of DNA found naturally in bacteria
plasmids can carry antibiotic resistance genes, genes for receptors, toxins or other proteins
plasmids can replicate separately from the genome of the organism
-must have origin of replication
plasmids can be engineered to be useful cloning vectors
vector is usually modified plasmid or bacteriophage
-has origin of replication
-carries cloned DNA
-must have restriction site(s)
multiple-cloning site useful
-selectable marker
ampicillin common
second marker helpful
distinguish recombinant
plasmids from intact vector
DNA cloning
isolate DNA
cut with restriction enzymes
join insert (DNA fragment) with vector (plasmid) to generate independently replicating recombinant molecule
introduce into host
Can place gene (insert) into high-copy-number vector
host bacteria will make large amounts of the protein
each gene copy can be transcribed and translated
genetic markers
Second marker: lac Z gene will be interrupted if plasmid takes up insert.
if insert, there will be no beta-galactosidase expression
so beta-galactosidase (lac Z) will not cleave X-gal.
when X-gal is cleaved, colonies are blue
multiple-cloning site in gene
insert interrupts lacZ′
intact vector —> blue colonies
recombinant molecule —> white colonies
applications of genetic engineering
Engineered plants and animals: transgenic
Protein production safer, more economical
-gene for human insulin cloned into bacteria
previously, insulin extracted from animals sometimes caused allergic reactions
-vaccine production: clone gene for specific proteins:
-vaccines for hepatitis B and cervical cancer
-foot-and-mouth disease of domestic animals
genetically engineered eukaryotes
yeasts can be engineered like bacteria; provide model
Ti plasmid from Agrobacterium tumefaciens used to generate corn, cotton, potatoes that produce Bt toxin from Bacillus thuringiensis
toxic only to insects and their larvae
soybean, cotton, corn engineered to resist biodegradable
plants with improved nutritional value
rice containing β-carotene; iron
recombinant DNA technology using Agrobacterium tumefaciens
example: insect resistant crops
Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties.
used as an insect-control strategy for many years in agriculture and gardening.
recently, plants developed (using Ti plasmid from Agrobacterium sp.) that express a recombinant form of bacterial protein, which may effectively control some insect predators.
Environmental issues associated with the use of transgenic crops have not been resolved.
lytic bacteriophages
lytic phage infections
lytic or virulent phages exit host
cell is lysed
productive infection: new particles formed
T4 phage (dsDNA) as model
entire process takes ~30 minutes
attachment
phage exploits bacterial receptors
genome entry
T4 lysozyme degrades cell wall
tail contracts, injects genome through cell wall and membrane
synthesis of proteins and genome
early proteins translated within minutes
nuclease degrades host DNA
protein modifies host’s RNA polymerase to
not recognize its own promoters
late proteins are structural proteins
(capsid, tail) produced toward end of cycle
assembly (maturation)
some components spontaneously assemble, others require protein scaffolds
release
lysozyme produced late in infection; digests cell wall
cell lyses, releases phage
burst size of T4 is ~200
temperate bacteriophages
option of lytic infection or
incorporation of DNA
into host cell genome
lysogenic infection
infected cell is lysogen
lambda (λ) phage as model
Temperate phages generally do not kill the bacterial cells they infect.
PROPHAGE: latent form of phage DNA that is present in lysogenic bacteria
Lambda phage
lambda (λ) phage: linear chromosome
complementary single-stranded overhangs at ends join inside host
resulting circular molecule either directs lytic infection or integrates into E. coli chromosome
phage enzyme integrase inserts DNA at specific site
site specific recombination
integrated phage DNA termed prophage
replicates with host chromosome
-can be excised by phage-encoded enzyme
-results in lytic infection
repressor prevents excision, maintains lysogenic state
DNA excision
-if DNA damaged (UV light exposure), SOS repair system turns on, activates a protease
-protease destroys repressor,
allows prophage to be excised,
enter lytic cycle
-called phage induction; allows
phage to escape damaged host
SOS repair system turns on, activates a protease:
RecA !!!!!!
SOS repair
During normal growth, the LexA repressor binds promoter region of SOS genes and prevents their expression.
When the cell senses DNA damage, the LexA repressor undergoes self-cleavage and the SOS genes are de-repressed.
RecA specifically binds single-stranded DNA (ssDNA),
promotes LexA cleavage (inducing the SOS response).
cI has structural similarity to lexA and in lysogens it is cleaved
-cleavage allows expression of lytic genes
In the SOS response, RecA cleaves LexA, removing SOS response repression.
cI also forms a repressor.
cI is the only factor needed to maintain the lysogenic state once it is established
cI forms a repressor.
cI is the only factor needed to maintain the lysogenic state once it is established.
RecA destroys cI.
PR and PL promoters are no longer repressed and switch on, and the cell returns to the lytic sequence of expression events.
Bacteriophages – consequences of lysogeny
lysogen immune to superinfection (infection by same phage)
Repressor maintaining integrated prophage also binds to operator on incoming phage DNA, prevents gene expression: immunity to superinfection
roles of bacteriophages in horizontal gene transfer
generalized transduction
-results from packaging error during phage assembly
-some phages degrade host chromosome; fragments can be mistakenly packaged into phage head
-these phages cannot direct phage replication cycle
termed generalized transducing particles
following release, can bind to new host, inject DNA
DNA may integrate via homologous recombination, replacing host DNA
any gene from donor cell can be transferred
Specialized transduction
-excision mistake during transition
from lysogenic to lytic cycle of
temperate phage
-short piece of flanking bacterial DNA
removed; piece of phage DNA remains
-excised DNA incorporated into phage
heads; defective particles released
-can bind to new host, inject DNA
-bacterial genes may integrate via
homologous recombination
-only bacterial genes adjacent to
integrated phage DNA transferred
filamentous bacteriophages
…either exclusively episomally replicating phage or temperate
phage that are chromosomally integrated, but can be
induced to start episomal replication
episome: a genetic element inside some bacterial cells, especially the DNA of some bacteriophages, that can replicate independently of the host and also in association with a chromosome with which it becomes integrated.
single-stranded DNA phages
used to produce only single-
stranded recombinant DNA
-look like long fibers
cause productive infections
-host cells not killed,
but grow more slowly
-M13 phage as model
attaches to protein on
F pilus of E. coli
single-stranded DNA genome enters cytoplasm
