Exam 3: Molecular Information Flow -- Replication, Transcription, Translation; Microbial Regulatory Systems; Genetics of Bacteria and Archaea; Viral Genomics (Bio 286 - Microbiology) Flashcards
biological information
genetic information contained within DNA (instructions necessary to build cells); information is INDEPENDENT OF THE MEDIUM upon which it is stored or encoded
nature of genetic material
MIESCHER – nuclein… GRIFFITH – transformation… AVERY/MACLEOD/McCARTY – transformation… HERSHEY and CHASE – blender experiment… CHARGAFF – the “rules” for nucleotide ratios
nucleosides
base + sugar
bases
adenine (A), thymine (T), guanine (G), cytosine (C)
nucleotides
base + sugar + phosphate
purines
adenine and guanine… two ring structures and larger
pyrimidines
cytosine, thymine, uracil… one ring structures and smaller
franklin and wilkins
X-ray diffraction pattern from a DNA smear looked like an X indicating that DNA had a helical orientation… Watson and Crick used this information of crystallography and biochemistry to figure out the structure
watson and crick’s model
postulated anti-parallel and double stranded molecule with bases on the inside… 3.4 nm per twist, 10 bp per twist… C pairs to G (with 3 hydrogen bonds) and A pairs to T (with 2 hydrogen bonds) [equal amounts of C and G and equal amounts of A and T/U]
two strands of DNA double helix are held together by
hydrogen bonds between nucleotide bases
Chargaff’s rules
purines match with pyrimidines: two purines would be too large and bulge and two pyrimidines would be too short to pair effectively… HYDROGEN BONDS FORMED BETWEEN NUCLEOTIDES
both DNA strands have same amount of information
bases in 1 strand are complementary to those in other strand
modern central dogma
replication -> transcription -> translation -> modification
meselson and stahl
observed intermediate and light DNA after two rounds of replication in light nitrogen… proved that DNA replication is semiconservative (with old DNA always remaining)
genome
complete cell DNA sequence
genotype
specific DNA sequence
phenotype
appearance and/or behaviour… a result of genotype and environment
prokaryotic genome
circular and haploid (mostly)
positive supercoiling
OVERWINDING the helix; tends to be performed in archaea
negative supercoiling
UNDERWINDING the helix; tends to be performed in bacteria
supercoiling
twists the DNA to condense it so it can fit inside the cell
type I topoisomerases
relieve torsional stress caused by supercoils
type II topoisomerases
introduce negative supercoils
archaeal topoisomerases
introduce positive supercoils
DNA replication
semiconservative replication; copies information to complementary strand; melt double-stranded DNA; polymerize new strand
oriC
where replication begins; DNA is opened at this site by helicases, where polymerization follows BIDIRECTIONALLY around chromosome
replication steps
- DNA helicase melts DNA… 2. Helicase recruits primase… 3. primer recruits clamp loader to each strand… 4. polymerase proceeds 5’ -> 3’ on each strand… 5. RNase H removes primers… 6. both forks move to ter sites
Replication Step 1. DNA helicase melts DNA
loader places HELICASE at each end of origin (oriC)… one helicase moves in each direction to copy genome
Replication Step 2. Helicase recruits primase
DNA POLYMERASE needs free 3’OH end… PRIMASE begins replication by forming a RNA primer with a 3’OH for DNA to attach
Replication Step 3. Primer Recruits Clamp Loader to Each Strand
clamp binds DNA polymerase III to strand
DNA polymerase III
performs most of DNA synthesis during replication
RNA synthesis does not require
primers
Replication Step 4. Polymerase proceeds 5’ -> 3’ on each strand
energy for polymerization comes from phosphate groups on recently added nucleotide… only proceeds 5’ -> 3’ because 5-phosphate of incoming nucleotide is attached to free 3’OH of growing DNA strand
Two Strands of a Replicating Fork
LEADING STRAND and LAGGING STRAND (OKAZAKI FRAGMENTS)
leading strand
follows helicase; has steady growth
lagging strand (okazaki fragments)
polymerase continues to previous primer…. clamp loader places primase on new site… DNA present in 1000 base pieces
Replication Step 5. RNase H removes Primers
one primer for each leading strand and many primers on lagging strands (one primer per okazaki fragment)… gaps filled in by DNA POLYMERASE I… DNA LIGASE seals nicks (creates phosphodiester bonds between nicked fragments of DNA– links okazaki fragments)
Replication Step 6. Both Forks Move to ter Sites
movement is simultaneous… opposite directions until both meet again at terminus… REPLISOMES ARE STATIONARY… DNA is threaded through replisomes
plasmids
EXTRACHROMOSOMAL PIECES OF DNA… LOW-COPY NUMBER (only one or two copies per cell)… HIGH COPY NUMBER (up to 500 copies per cell, divide continuously, randomly segregate)
plasmid replication
BIDIRECTIONAL replication (similar to chromosomal replication) or UNIDIRECTIONAL replication (“rolling circle” replication, similar to phages)… starts at nick bound by RepA protein -> provides 3’OH for replication -> helicase moves around plasmid repeatedly
plasmid genes
advantageous under special conditions… ANTIBIOTIC-RESISTANCE genes, genes encoding resistance to toxic metals, genes encoding proteins to METABOLIZE rare food sources, VIRULENCE genes to allow pathogenesis, genes to allow SYMBIOSIS… contain genes that are not essential for cell growth/replication
gene
functional unit of genetic information
GTP (guanosine triphosphate)
provides energy for translation
transcription steps
- Initiation… 2. Elongation… 3. Termination
transcription step 1. initiation
bind polymerizing machine, first monomer to template… involves DNA polymerase, RNA polymerase, and ribosome
transcription step 2. elongation
read template, add next monomer… DNA, RNA, Protein
transcription step 3. termination
release machinery and completed product
replication template
DNA
replication product
DNA
replication monomers
dA, dC, dG, dT
replication enzyme
DNA polymerase III
replication direction
5’ -> 3’
replication start
oriC
replication end
ter
transcription template
DNA
transcription product
mRNA
transcription monomers
A, C, G, U
transcription enzyme
RNA polymerase
transcription direction
5’ -> 3’
transcription start
promoter
transcription end
terminator
translation template
mRNA
translation product
protein
translation monomers
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y
translation enzyme
ribosome
translation direction
N -> C
translation start
shine dalgarno (RBS)
translation end
stop codon
translation enzyme
ribosome
translation enzyme
ribosome
RNA polymerase
4 proteins in one complex (core complex – α, β, β’, ω) and becomes the holoenzyme when σ joins the complex… binds DNA, reads sequences, and polymerizes RNA
sigma (σ) factor
guides RNA polymerase to target DNA sequence (PROMOTER) to start transcription
the new RNA molecule made from the DNA template
is antiparallel and complementary to template
transcription elongation
core polymerase adds RNA to 3’ end (energy for base addition comes from base)… the added base is complementary to template strand and mRNA has the same sequence as non-template
RNA polymerases do not need
primers
sigma 35 (σ^35) factor
heat shock response factor
transcription rho-dependent termination
Rho (ρ) factor binds to mRNA… slides along mRNA up to polymerase… breaks polymerase, mRNA off of DNA
Rho (ρ) factor
protein serving to terminate transcription in bacteria
Rho-independent termination
series of U residues downstream of pause site… DNA-RNA UA base pairs are least stable… even less stable of polymerase is stalled… mRNA breaks off of DNA and polymerase released
termination of RNA synthesis is ultimately determined by
specific nucleotide sequences on template strand of DNA
operon
allows coordinated expression of multiple related genes in prokaryotes
mRNA
messenger codes for peptides
rRNA
ribosomal structure and function
tRNA
transfers amino acids to ribosome during translation… adapters between nucleic acid and proteins
snRNA
small nuclear (splicing of message)
miRNA
microRNAs (regulate expression)
CRISPR
prokaryotic “immune system” – RNA based
stop codons
UAA, UAG, UGA
genetic code
consists of nucleotide triplets called CODONS– 61 specify amino acids (START CODONS – aka sense codons // STOP CODONS – aka nonsense codons)… code is degenerate/redundant (multiple codons can encode same amino acid)… code operates universally across species
tRNA structure
specific shape with a 3-base anticodon arm (which base pairs to codons in mRNA) and an amino acid attachment site (proteins use the aminoacyl-tRNA transferase to add amino acid)
ribosome
PROTEIN POLYMERASE… very large molecular machine with 2 subunits, 52 proteins, and 3 rRNAs
ribosome active site
70S ribosome harbors three binding sites for tRNA – A site, P site, and E site
A (acceptor) site
binds incoming aminoacyl-tRNA
P (peptidyl-tRNA) site
harbors the tRNA with growing polypeptide chain
E (exit) site
binds a tRNA recently stripped of its polypeptide; where tRNA is released from ribosome
translation step 1. initiation
performed only once… IF1 and IF3… starts at the shine dalgarno sequence… 16S rRNA… IF2 and tRNA-FMET… GTP hydrolyzed
translation step 2. elongation
EF-Tu and tRNA… enter A site… transpeptidation… EF-G… translocation…. 3 GTP per amino acid added
initiation of translation is prevented by
tetracyclines
elongation of translation is prevented by
mycins (streptomycin, erythromycin)
translation step 3. termination
stop codon encountered… TAA, TAG, TGA… RF1 and RF2… peptide released… RF3 ejects RF1 and RF2… RRF dissociates subunits
coupled transcription and translation is performed by
archaea, bacteria
coupling of transcription and translation
TRANSCRIPTION CREATES mRNA –> MULTIPLE mRNAs MADE FROM A SINGLE GENE…. RIBOSOMES BIND mRNA –> WHILE mRNA IS STILL BEING CREATED… multiple proteins made rapidly from each mRNA, which is the advantage of not having a nucleus
coupling of transcription and translation cannot occur in eukaryotes because
ribosomes are outside of nucleus
eukaryotic expression
partitioning of steps… RNA splicing (to remove INTRONS from EXONS)… no operons are present… multifunctional proteins are formed… modular approach
exons
protein coding regions of eukaryotic genes
eukaryotic transcription occurs in
nucleus
RNA splicing
removes introns from the primary RNA transcript to form the mature mRNA of exons
protein modification
enzymes modify translated proteins… fMet removed from N-terminus… small groups added to amino acids (PHOSPHORYL, METHYL, or ADENYLATE groups added)… protein may be cleaved or refolded by helping enzymes
protein structure is determined by
amino acid sequence (causing spontaneous folding) and CHAPERONES (refolds denatured proteins using ATP)
transcription of chaperones is greatly accelerated when
a cell is stressed by excessive heat
protein transport
many bacterial proteins reside in cytoplasm while others are targeted to other sites (plasma membrane, periplasm, gram (-) outer membrane, secreted outside of bacterium)… SIGNAL SEQUENCE TARGETS PROTEINS FOR TRANSPORT
type II secretion system
N-TERMINAL AMINO ACIDS bound by SecB… targets ribosome to SecA complex… energy dependent efflux to periplasm… moves across one membrane (to periplasm)
type I secretion system
secretes protein out of bacterium… many other secretion systems known… moves across two/both membranes to leave cell
proteasomes
degrade proteins that are flagged as damaged
ubiquitin
adds signal to proteins/tag causes degradation of proteins (by signaling that protein is damaged/nonfunctioning)
regulating gene expression
microbes RESPOND to changing environment by altering growth rate, proteins produced, and behaviour… so they must be able to sense their environment, needing RECEPTORS to transmit information to chromosome… and when they need to change enzyme function they do so through TRANSCRIPTIONAL, translational, and post-translational mechanisms
two component regulatory systems are useful for controlling gene expression in response to environmental signals because
phosphorylation is a permanent change so genes are always turned on after signal
two component signal transduction
SENSOR KINASE protein in plasma membrane, which binds to signal (can respond to membrane fluidity)… and cytoplasmic RESPONSE REGULATOR, which alters transcription rate of chromosomal genes (specific to phosphorylation)
sensor kinases that respond to extracellular signals transfer
histidine signal to cytoplasmic membrane machinery by typically phosphorylating residues
organism that would likely harbor the most two-component regulatory systems
bacterium occupying a heterogeneous niche with high nutrient mixing
sigma factor initiates transcription by RNA at
PROMOTER
regulatory proteins
bind to OPERATOR sequences
activators
bind to operator and increase strength of gene’s transcription
repressors
bind to operator and lower strength of gene’s transcription
gel mobility shift
-DNA moves through a gel faster when it is not bound to protein (supershift) and less shifting with less competing factors
-Gel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein.
E.coli Lac operon
LACTOSE (milk sugar) is used for food, but cannot pass through plasma membrane – LACTOSE PERMEASE allows entry, with PMF used to bring lactose inside cell… must be converted to glucose to be CATABOLIZED… beta-galactosidase (lac-Z) converts lactose to glucose…. operon consists of lacZ, lacY, and lacA
two regulators controlling one operon must respond to different signals
which enables both to control operon differently
operon
group of coordinately expressed and regulated genes associated with common purpose
lacZ
gene encodes beta-galactosidase
lacY
gene encodes lactose permease
lacA
gene encodes a transacetylase
E.coli lac operon (with no lactose)
REPRESSOR protein LacI blocks transcription… repressor binds to operator and blocks sigma factor from binding promoter
lactose operon on with glucose and on with lactose occurs in
a Lac knock-out mutant
mutation in gene encoding the lactose repressor (LacI) that prevents lactose from binding to the LacI protein would result in
constant repression of lac operon in presence of lactose
E.coli lac operon (with lactose)
repressor responds to presence of lactose… binds inducer (ALLOLACTOSE) or DNA (not both) and adds lactose, causing the REPRESSOR TO FALL OFF OPERATOR
catabolite repression leads to
leads to a bacterium using up glucose before using any lactose (choosing its carbon source)
diauxic growth
biphasic curve of a culture growing on two carbon sources
catabolite repression
operon enabling catabolism of one nutrient is repressed by presence of a more favorable nutrient (commonly GLUCOSE– the easiest sugar to catabolize)…. glucose is transported using a PHOSPHOTRANSFERASE system… presence of glucose affects an internal signal (cAMP)…. IIA^Glc inhibits ADENYLATE CYCLASE and reduces internal cAMP pool… ie, HIGH GLUCOSE -> LOW cAMP
cAMP
secondary messenger molecule formed by adenylate cyclase
adenylate cyclase
turns off with high glucose… synthesizes cyclic cAMP from ATP, which is involved in catabolite repression
cAMP affects transcription
maximum expression of lac operon requires presence of cAMP and cAMP RECEPTOR PROTEIN (CRP)… CRP is an INDUCER of lac operon
inducer exclusion
glucose transport also inhibits lactose transport – IIA^Glu uncouples LacY [when glucose is present, LacY is off]… few transporters that are present are no longer functional
arabinose operon control
ara operon… AraC acts as REPRESSOR to block transcription –> when arabinose is added, CONFORMATION IS CHANGED so that it now acts as an ACTIVATOR, stimulating binding of RNA polymerase
Trp Operon Transcription
Trp operon contains 5 genes to make TRYPTOPHAN… is ONLY EXPRESSED IN ABSENCE OF TRYPTOPHAN… OPPOSITE OF LAC REPRESSOR… Trp APOREPRESSOR must bind to tryptophan in order to bind the operator as the HOLOREPRESSOR
TrpR
exhibits negative repression
attenuation
type of regulation that can control transcriptional activity exclusively; regulatory mechanism in which translation of a LEADER PEPTIDE affects transcription of a downstream structural gene
attenuation does not depend on
conformational change in protein/enzyme structure to change activity
attenuation of Trp operon
ATTENUATOR region of trp operon has 2 trp codons and is capable of forming stem-loop structures
mechanism of attenuation in high tryptophan levels
- ribosome translates through trp codons and encounters translation stop codon… 2. ribosome stops, covering mRNA regions (and polymerase continues to transcribe regions 3 and 4— 3:4 TERMINATION LOOP FORMS– ATTENUATOR STEM LOOP)… 3. 3:4 loop binds RNA polymerase and causes its release before reaching trpE
mechanism of attenuation in low tryptophan levels
- ribosome translates leader… 2. scarce tRNA*p makes ribosome stall at Trp codons and polymerase continues through attenuator… 3. stalled ribosome covers region 1, allowing 2:3 STEM LOOP (ANTI-ATTENUATOR STEM LOOP) to form, where the less energetically favorable 3:4 loop cannot form…. 4. polymerase transcribes TrpE
riboswitches
METABOLITE DIRECTLY BOUND BY mRNA… induced CONFORMATIONAL CHANGE… results in either 1) TRANSCRIPTION TERMINATION, 2) RIBOSOME EXCLUSION, or 3) mRNA DEGRADATION
control of bacteriophage lambda– lytic cycle
phage quickly replicates and kills host cell… generally lytic when host conditions are good or conditions are very bad (ex: cell damaged)
control of bacteriophage lambda– lysogenic cycle
phage is quiescent… may integrate into host cell genome… replicates only when host genome divides…. generally lysogenic in moderate cell conditions… phage can reactivate to become lytic and kill host
λ cI repressor prevents lytic cycle
binds to Or operator to block Pr promoter (prevents synthesis of cro protein)… binds to block Pl promoter (prevents synthesis of downstream lytic proteins)
Cro protein prevents synthesis of cI
represses Prm promoter (blocks synthesis of cI)… activator for Pl promoter (stimulates lytic protein synthesis)
more cI
LYSOGENY
more Cro
LYSIS
cro (regulatory protein)
favors the lytic cycle in lambda phage
lysis vs lysogeny for bacteriophage lambda
depends on multiplicity of infection (MOI) – high MOI -> cII made, stimulating cI synthesis (turns off lytic and turns on lysogeny)… low MOI -> cII degraded by cell protease…. stationary phase -> cII accumulates, cI made, so lysogeny is favored
logarithmic growth favors
lysis
ways eukaryotic gene regulation differs from prokaryotes
most genes are CONTROLLED INDIVIDUALLY… presence of INTRONS… use of DIFFERENT RNA POLYMERASES… use of generalized and specialized transcription factors… bind to regulatory DNA sequences called ENHANCERS and SILENCERS
three codons in the genetic table code for
STOP
homoserine lactones are involved in
quorum sensing in Gram Negative bacteria
when regulatory protein binds positively (on) and has catabolic induction (on) when substrate is present
CRP
when regulatory protein binds negatively (off) and has catabolic induction (on) when substrate is present
LacI
when regulatory protein binds negatively (off) and has anabolic repression (off) when substrate is present
TrpR
alternative sigma factors can be controlled by
ALTERED TRANSCRIPTION, TRANSLATION, PROTEOLYSIS, and ANTI-SIGMA FACTORS
alternative sigma factors examples
HEAT SHOCK, SPORULATION, or FLAGELLA SYNTHESIS
RpoH sigma 32
heat shock response genes
functions of heat shock proteins
degradation of denatured proteins; responding to exposure to high levels of ethanol; prevention of inappropriate protein subunit aggregation
heat shock respiration
1) at 30 degrees C, RpoH is transcribed but the secondary structure of mRNA hides the ribosome binding site; very little σ^H is made…. 2) DnaK-DnaJ-GrpE chaperones shunt σ^H to degradation… 3) at 42 degrees C, the secondary structure melts and ribosomes can more easily bind and translate σ^H… 4) at 42 degrees C, proteins denature from their native folded forms to their unfolded forms. the unfolded forms are bound by DnaK-DnaJ-GrpE (meanwhile, chaperones refold denatured proteins)…. 5) freed from DnaK-DnaJ-GrpE, σ^H is not degraded and can drive expression of heat-shock genes
components of two-component signal transduction pathway
histidine kinase and response regulator
phosphorylation
method of protein modification in a two-component signal cascade
endospore formation
sigma cascade… PRO-SIGMA PROCESSING used by mother cell and ANTI-SIGMA FACTORS used by endospore… cross talk between mother cell and endospore (mediated by sigma factor through use of protease)…. coordination of cell activities
σ^F and σ^G
sequestered as anti-sigma factors used by endospore
small regulatory RNA (sRNA)
found within bacterial intergenic regions and REGULATE THE TRANSCRIPTION or STABILITY OF mRNAs… ANTISENSE nature of sRNA allows these molecules to bind mRNA… can either STABILIZE the target mRNA or MAKE IT SUSCEPTIBLE TO DEGRADATION… exert their effects by base pairing with other RNA molecules that have regions of complementary sequence
DNA rearrangement
some microbes use gene regulation to periodically change their appearance in a process called PHASE VARIATION (such as with flagellar proteins in Salmonella enterica)… OCCURS BY GENE INVERSION…. invertible PROMOTER SWITCH regulates two genes encoding different flagellin types, with expression depending upon its orientation
phase variation of flagellar proteins (flagellin H1 or H2) in Salmonella enterica
- promoter drives transcription of FijB and FijA… 2. salmonella expresses H2 flagellin… 3. FijA expresses FliC… 4. Hin recombinase is made and binds to hix sequences… 5. Hin dimer brings Hix regions together and then breaks/rejoins ends to invert whole sequence… 6. promoter is in wrong orientation… 7. after DNA inversion, Salmonella expresses H1 flagellin
riboswitches
possibly one of earliest forms of metabolic regulation that evolved
chemotaxis
behaviour in which motile bacteria swim towards favorable environments (CHEMOATTRACTANTS) or away from unfavorable environments (CHEMOREPELLANTS)… occurs through use of a modified two component system
direction of flagella motor rotation determines type of movement
counterclockwise rotation results in smooth swimming/running and clockwise rotation results in tumbling
default setting for flagella rotation in E.coli is
counterclockwise
when conditions become favorable, flagella rotate
counterclockwise
methyl-accepting chemotaxis proteins (MCPs)
sensitivity set by methylation; transmit a signal to regulate a switch
when MCP proteins have methyl groups added
they become less sensitive (the more methyls added, the more decreased sensitivity)
nitrogen regulation
glutamine synthase (GlnA) uses nitrogen (NH4+) to convert glutamate into glutamine
excess glutamine
excess nitrogen present
excess glutamate
scarce amount of nitrogen present
GlnA regulation
GENETIC CONTROL – two component (glutamine synthetase makes glutamine when there are low levels of nitrogen– excess glutamate)… BIOCHEMICAL CONTROL – post-translational (glutamine synthetase is inactivated when AMP is added to GlnA in high levels of nitrogen– excess glutamine)
regulation of enzyme activity occurs
posttranslationally
quorum sensing
bacteria respond to CELL DENSITY… discovered in Vibrio fuscheri (a BIOLUMINESCENT bacterium that colonizes the light organ of Hawaiian squid)
Quorum Sensing Mechanism
induction requires the accumulation of a secreted small molecule called an AUTOINDUCER… at a certain extracellular concentration, the secreted autoinducer is detected, and the signal then alters gene expression (for bioluminescence, or virulence)
the greater the cell density
the more autoinducer secreted
transcriptome
constitutes all of a cell’s mRNA MOLECULES; continually changes in response to a changing environment
proteome
constitutes all of a cell’s PROTEINS; continually changes in response to a changing environment
DNA microarray (gene array)
can simultaneously examine the expression of every gene in the cell… uses a DNA MICROCHIP (DNA fragments from every ORF in a genome are affixed to separate locations on a solid support surface, producing a grid/array)… used to analyze RNA extracted from microbes grown under two different environmental conditions (COMPLEMENTARY DNA (cDNA) is made first)
gene chips
used in microarray… a technique to study transcriptomics
two dimensional gel electrophoresis
used to view and capture fluctuations in proteome… the first dimension separates proteins by ISOELECTRIC POINT and the second dimension further separates proteins by MOLECULAR WEIGHT
2D gels
technique used to study proteomics
identifying proteins from a 2D gel
- proteins extracted from bacterial culture… 2. 2D electrophoresis… 3. spots of interest cut out of gel… 4. protein spot isolated… 5. protease added to digest protein… 6. peptides produced…. 7. analysis by mass spectrometry… 8. mass calculations provide molecular weight of each peptide… 9. protein identified by sum of its peptide masses
bacterial chromosome
repository of most genes in cell… genotype affects cell’s phenotype… must be transferred vertically to progeny, but could also be transferred horizontally… important for rapid dissemination of favorable traits (ex: drug resistance)
DNA sequence is not static
can be altered through mutations of single bases, large deletions, or large insertions of sequence (transferred from other species)… maintained via interaction with environment (with survival determined by having appropriate genes for specific environment
plasmids
small circular DNA…. autonomously replicating… multiple copies per cell… can be transferred between cells… associated with antibiotic resistance (R)… commonly used in molecular biology
transforming principle
GRIFFITH’s experiments with infections of mice using strains of streptomyces (ROUGH and SMOOTH colony types) [with the smooth phenotype due to capsule protection]
AVERY, MacLeod, McCarty
extended Griffith’s transforming principles– fractionated killed cells, using enzymes to destroy factors: protease and RNase had no effect and permitted transformation to still occur, but removing DNase cause no transformation to occur, demonstrating that DNA is needed for transformation – DNA IS THE TRANSFORMING MATERIAL
competence
ability to TAKE UP EXOGENOUS DNA; requires special proteins such as cell wall autolysin… some bacteria naturally contain this ability while others need to be coaxed (such as with calcium chloride, heat shock, and electroporation)
genetic transformation
competent cells (natural or artificial) + NAKED DNA taken up, incorporated, and expressed
in conjugation, the donor cell
survives the genetic transfer
F (fertility) plasmid
contains a set of genes that encode for the pili proteins that are essential in conjugative transfer of DNA
F+ strains
have the F factor as a plasmid
conjugation
plasmid-directed transfer REQUIRES CELL CONTACT
if F plasmid is not integrated into chromosome,
cell surface receptors change, preventing uptake of more plasmids through conjugation
Hfr strains
(high frequency of recombination); INTEGRATED F FACTOR… conjugal transfer… chromosomal genes introduced/incorporation of new genes into chromosome… this state is most similar to lysogeny
episomes
plasmids that can incorporate
transduction types
generalized and specialized
generalized transduction
involves a LYTIC PHAGE… infection as usual -> mistaken packaging of host gene -> defective gene -> the defective phage binds -> inserts DNA -> no new viruses made -> incorporation into genome… so, the host DNA is packaged into a bacteriophage
specialized transduction
involves a LYSOGENIC PHAGE…inserts as prophage -> aberrant excision -> picks up adjacent host gene -> defective phage…. MINIMAL AMOUNT OF GENETIC INFO NEEDED: ALT REGION, COS SITE, and HELPER PHAGE
lysogeny carries a strong selective advantage for the host cell
because it confers resistance to infection by viruses of the same type
defense against transferred DNA
bacteria cut entering DNA to pieces– cut at specific RESTRICTION SITES… bacteria add METHYL GROUPS to DNA – prevents restriction at those sites, added as cell replicates chromosome… entering DNA is destroyed– unless coming from a similar species or has methyl groups protecting DNA
transformation distinction
needs naked DNA
conjugation distinction
needs cell contact
transduction distinction
involves bacteriophage
methods of introducing foreign DNA into a recipient
transformation, conjugation, transduction
DNase
will inhibit transformation
0.2 micrometer membrane filter
will disrupt conjugation
recombination
entering DNA replaces chromosomal DNA… if sequence is overall similar, DNA enters via transformation, conjugation, or transduction – replaces variable-sized section of DNA, or USED TO REPAIR DAMAGED DNA… requires specific recombination proteins– RecA, RecBCD, and RUVAB
RecA
catalyzes integration of linear transforming DNA into the chromosome
mutations
mistakes made during replication or damage to DNA
mutations where the wrong bases are incorporated
TRANSITION – little to little bases, most common // TRANSVERSION – little to big or big to little bases
mutants
organisms containing mutations
mutagens
increase error rate or mutations
auxotroph
mutant strain with an additional nutritional requirement for growth
mutagens cause mutations
electromagnetic radiation (X-rays, gamma rays, UV light)… spontaneous tautomers during replication… chemicals (analogs of bases, base-modifying chemicals (nitrosoguanidine, nitrous acid), intercalators insert between bases (causing frameshift mutations)
point mutations
a single base is altered in the sequence; includes silent, missense, and nonsense mutations
silent mutation
no change in amino acid sequence from change to codon sequence; most tolerated mutation
missense mutation
a change in amino acid sequence to another amino acid sequence due to change to the codon sequence
nonsense mutation
a change in amino acid sequence to a STOP sequence due to change to codon sequence; least tolerated/worst phenotype
5-Bromouracil mutagenesis
A base becomes a G base – TRANSITION MUTATION
measuring mutagen strength
AMES TEST created by Bruce Ames, uses Salmonella typhimurium to test mutagens– His- mutant strain grown in absence of histidine and loo for reversion to His+
frame shift mutations
genetic recombination involving insertion sequence– adding/deleting 1 or 2 bases knocks the sequence out of frame so that the same protein is no longer made
cystic fibrosis
results from in-frame deletion
potential reading frames
three reading frames in forward direction and three reading frames in reverse direction due to the reading of triplet codons
DNA repair mechanisms
MISMATCH REPAIR (mispaired base cut out of strand, strand without methyl group is newer and assumed to be in error)… THYMIDINE DIMERS (induced by UV, cut out by UVrAB complex)… damaged bases (excised by specific enzymes, replaced by DNA polymerase I)… RECOMBINATIONAL REPAIR (occurs just after strand has replicated, undamaged strand is copied and replaced damaged strand, catalyzed by RecA recombinase)… SOS REPAIR (extensive DNA damage inactivates LexA, activation of many repair genes, rapid polymerization of DNA, error-prone but better than no repair)
horizontal gene transfer
movement of genes between cells through transformation, conjugation, or transduction
effects of gene transfer
spreads useful genes among bacteria– antibiotic resistance genes (spread wherever antibiotics are overused), pathogenicity islands (encode genes for cell to act as pathogen), genes to degrade special metabolites
a gene located on a chromosome would be
least likely to be transferred
evolutionary relatedness of life
archaea share many genes with bacteria and share other genes with only eukaryotes (midway between bacteria and eukarya)… difficult to discern bacterial history (genes in one cell may not have been inherited from parents as it could be obtained instead from other bacteria and bacterial species are related through lateral transfer as well as parentage)
transformation requirements
competence, naked DNA, any gene
conjugation requirements
plasmid with a pilus, direct contact, bias for certain genes
transduction requirements
phage
generalized transduction requirements
lytic, any gene
specialized transduction requirements
lysogenic, biased for genes
mobilome
total of all mobile genetic elements in a cell’s genome; includes SELF SPLICING RNA (enzymatic genetic elements), TRANSPOSONS (mobile genetic elements), PLASMIDS (autonomous genetic elements), and VIRUSES (infectious genetic elements)
how extensive the mobilome is
all (or very nearly all) cells have mobile genes– 50% OF HUMAN GENOME IS MOBILE GENETIC ELEMENTS… 90% of wheat genome, but 2% of E.coli genome (but majority of plasmids)
group II introns
catalytic genes… LARGE RIBOZYMES and SELF SPLICING… found in all domains, forms a lariat, and the ancestor of mRNA splicing
transposition
site-specific recombination event
kinds of transposable elements
DNA TRANSPOSONS (insertion sequences, transposons, and conjugate transposons) and RETROTRANSPOSONS (retrons (msDNA), SINE, LINE, and LTR)
insertion sequences (IS elements)
type of DNA transposon– INVERTED TERMINAL REPEATS… TRANSPOSASE… REPLICATIVE OR NON-REPLICATIVE TRANSPOSITION
transposons
type of DNA transposon– COMPOSITE (capture an intervening gene– is between two IS elements) or COMPLEX (gene with element– within an IS element)
enzyme transposase may be coded for by insertion sequences
on a chromosome, phage, or plasmid
transposase
enzyme used to mobilize insertion sequences in bacteria
conjugative transposons
type of DNA transposon– SXT encodes sulfa-drug resistance, mobile element (transposon), excise to circular form, encodes genes for conjugal transfer
genomic islands
provide evidence for horizontal gene transfer; altered G and C percent composition; includes PATHOGENICITY ISLANDS, SYMBIOSIS ISLANDS, and FITNESS ISLANDS
retrons
type of retrotransposon– ms-DNA – SATALLITE DNA IN PROKARYOTES… widely distributed in bacteria and archaea, made by reverse transcriptase.. BOTH ssDNA AND ssRNA base paired together… not yet proven to be mobile and have no known function
SINE
type of retrotransposon – SHORT INTERSPERSED ELEMENTS… <500 base bairs, short… 1,500,000 in human genome (11%)– MOST NUMEROUS IN GENOME… RNA polymerase III genes… Alv SEQUENCES… no RTase gene… mobilizable… COMPOSITE SINES
LINE
type of retrotransposon– LONG INTERSPACED ELEMENTS… up to 9,000 base pairs… 500,000 in human genome (17%)… RNA polymerase II genes… code for RTase… replicates transposition
LTR
type of retrotransposon– LONG TERMINAL REPEATS… is missing Env (but has pol and Gag retroviral components)… around 500,000 in human genome (8%)… RTase gene… SIMILAR TO RETROVIRUSES… LACK ENVELOPE PROTEINS
plasmids
RETROPLASMID (rare, found in some fungal mitochondria); copy number variation (1 to hundreds); EPISOMES; CONJUGATION
addiction modules
toxin-antitoxin set on plasmid… ANTITOXIN IS UNSTABLE… if the plasmid is lost from the cell: protease destroys antitoxin, toxin is activated, and the cell dies -> PLASMID ENSURES THAT CELL DOES NOT LOSE THE PLASMID (the cell becomes “addicted” to having the toxic plasmid)
bacteriophage
CIRCULAR genomes… ROLLING CIRCLE replication… LYTIC phage… TEMPERATE phage… requires host cell machinery… CAN MOBILIZE HOST GENES
herpes virus (class I)
binding -> membrane fusion -> inject through nuclear pore (viral genes get inside nucleus) -> rolling circle replication -> early genes (decision) -> late genes (assembly) -> acquire envelope from nuclear membrane (or ER or golgi) -> exocytosis
parvovirus (class II)
ssDNA…. use host DNA polymerase… ROLLING HAIRPIN mechanism to deal with ends
most RNA viruses replicate and assemble in the
cytoplasm
EHDV (class III)
double stranded RNA… binding and viropexis… remains in cytoplasm (never uncoats)… viral proteins from virus factories… negative strand RF… assemble and synthesize positive strand
poliovirus (class IV)
binding -> viropexis -> ER vesicles form -> viral RDRP made (RNA dependent RNA polymerase) -> minus strand RF -> 50,000 positive strands -> late genes -> assembly -> exit (By lysis)
influenza virus (class V)
bind via HA -> viropexis -> membrane fusion -> viral RDPR pre-made -> enter nucleus -> minus strand RF -> plus strand progeny made by viral polymerase -> cap snatching -> exit via budding (NA)
influenza variation
segmented genome… ANTIGENIC DRIFT (slow, accumulation of mutations over a season) or ANTIGENIC SHIFT (reassortment/new combination of strains to form new strains with various affects– rapid appearances of novel strains)
(antigenetic) genetic shift
recombines gene fragments during infection
HIV (class VI)
bind CD4/CCR -> reverse transcriptase (takes RNA to make DNA) [ +RNA -> -DNA -> dsDNA ] -> circularize -> integrate -> expression -> assembly -> budding
HBV (class VII)
dsDNA… bind and entry -> repairs in nucleus -> mRNA -> assembly -> reverse transcriptase [+RNA -> -DNA -> sdDNA] -> exit
dependent viruses
VIROPHAGES OF MIMIVIRUS (mimicking viruses , obligate parasites)… DEFECTIVE VIRUSES (unable to cause an infection by themselves, requiring another virus for replication)
hepatitis D
defective virus… needs HBV envelope protein… cannot package its core by itself
