MCBL Final Exam

conjugation

"bacterial sex" - mating for gene transfer type IV secretion system = sex pilus The recipient cell receives a copy of the F factor and becomes F+, gains ability to transfer DNA

transposons

"jumping genes" discovered by Barbara McClintock in corn (Nobel Prize 1983) segments of DNA can move from one plae in genome to another - transposition used in molecular genetics to generate mutants (biological mutagens)

superinfection immunity

1. CI suppresses lambda gene expression 2. att site is unavailable

SOS response

1. LexA repressor binds to target genes. SOS system is off 2. DNA damage causes accumulation of ssDNA, SOS system ON 3. RecA binds ssDNA, coprotease is activated, LexA is autodigested RecA cleaves lexA (suppressor of SOS), SOS genes are de-repressed for DNA repair UmuDC: DNA polymerase that repairs DNA rapidly but without proofreading, generating numerous mutations (error prone repair)

transcription activation

1. activator binds ligand; complex binds the regulatory sequence and activates target gene; removing ligand stops transcription 2. removing the ligand stops transcription 3. target gene is not expressed

two step replication of T4 DNA

1. bidirectional replication produces several copies of linear genomes with sticky 3' ends 2. long linear concatemer is formed through homologous recombination 3. concatemer is cleaved as DNA is packaged into capsid heads 4. headful packaging - individual genomes are cut at positions slightly more than one complete set of T4 genes 5. each phage head contains the same genetic information with different regions at ends - circularly permuted 6. generate terminal redundant ends

glucose inhibits lactose metabolism

1. glucose is transported through the phosphotransferase system 2. protein IIA obtains a phosphoryl group from PEP and passes it to IIB and then to glucose 3. unphosphorylated IIA directly inhibits the permease activity of lacY (inducer exclusion) 4. phosphoyrlated IIA (glucose is absent) activates adenylate cyclase which catalyzes the reaction to produce cAMP 5. lac operon is not activated due to no lactose transport and low cAMP levels

low tryptophan levels

1. ribosome translates through trp codons and encounters translation stop codon 2. ribosome stops, covering mRNA regions 1 and 2. polymerase continues to transcribe regions 3 and 4. 3:4 termination loop forms. 3. 3:4 loop binds RNA polymerase and causes its release before reaching trpE. majority of ribosomes do not stall at the two trp codons but reach the translation stop codon of the trpL MRNA the 3-4 attentuator loop is formed to terminate the transcription together with a U-rich region (trp structural genes are not transcribed) small percentage of ribosomes will stall at the trp codons, trp structural genes are transcribed in a low level - fine tuning the production of tryptophan

CRISPR-mediated DNA disruption

1. spacer acquisition (virus invades cell. new spacer is derived from virus and integrated into CRISPR sequence. 2. crRNA processing (CRISPR RNA is formed) 3. target invading DNA (CRISPR RNA guides molecular machinery to target and destroy viral genome)

replication of phage lambda

2 step replication 1. omega replication (circle to circle) to generate several copies of the circular viral genome. initiates at ori site, bidirectional 2. each circular genome is replicated through rolling circle replication. one strand is nicked at cos site, produces long chain of concatenated lambda genomes individual genome is separated by terminase at the cos site for packaging as linear DNA

Low MOI

CII degraded by protease Cro accumulates and activates gene transcription from PL and PR - lytic cycle starts CI is not transcribed

high MOI

CII proteins accumulate due to high CII vs E coli protease ratio CIII is transcribed from PL - inhibits E coli protease to further promote CII accumulation CII activates cI transcription and transcription of int (encodes the integrase) CI represses the transcription of all other genes, maintains its own transcription from PRM

high frequency recombination (Hfr)

F factor integrated into bacterial chromosome via recombination Hfr = bacteria that carry an F factor in chromosome (donor cells - male) fragment of chromosomal DNA is transferred during conjugation - integrated into genome of recipient (F-) cell by recombination. recipient cell remains F-. Chromosomal genes transferred in order depending on their distance to oriT (gene mapping)

sRNA regulation - iron metabolism

Fur: represses the transcription of enterochelin and rhyB - Fe(2+) is the corepressor ent: encodes siderophore that promotes iron uptake rhyB: encodes a sRNA that regulates the sucCDAB operon which encodes enzymes that require iron to function

rolling circle replication

Replication of circular DNA that is initiated by a break in one of the nucleotide strands, producing a double-stranded circular DNA molecule and a single-stranded linear DNA molecule, the latter of which may circularize and serve as a template for the synthesis of a complementary strand. okazaki fragments are synthesized last piece of DNA that is transferred = oriT

error prone repair

SOS response extensive DNA damage (accumulation of single stranded DNAs) activates RecA (coprotease) RecA cleaves LexA (SOS suppressor) SOS genes are de-repressed for DNA repair UmuDC: DNA polymerase that repairs DNA rapidly but without proofreading, generating numerous mutations

promoter

The binding site of RNA polymerase into an operon

high concentration of tryptophan

TrpR is activated, no transcription, no translation

competence

ability of cells to take up foreign DNA disruption of DNA replication increases bacterial competence gram negative bacteria do not use transformasome or CF. some are always competent. cell surface-anchored pilli bind extracellular DNA for uptake. physiological states of bacteria affect competence (gram neg) ie stresses (starvation, temperature)

cAMP

activator complex CRP acts as an activator only when bound to cAMP (inducer), which accumulates when a cell is starved for carbon cAMP-CRP complex binds to a DNA region upstream of the lacZYA promoter - CRP then directly interacts with the alpha subunit of RNA polymerase and increase the rate of transcription initiation only induces lac operon transcription in presence of lactose when Lacl is active, lac operon is turned off even with CRP-cAMP is present

RpoS

an alternative sigma factor for stationary phase can only accumulate when DsrA is present

allolactose

an inducer of lacZYA reduces lacL affinity for LacO and transcription of the operon occurs

methylation enzymes

bacteria adds methyl groups to its own DNA so that restriction sites are protected if not methylated, foreign DNA will be digested unless it comes from a similar species or has the restriction sites protected using other mechanisms

transduction

bacteriophage serves as carrier of bacterial DNA (errors during viral packaging) sometimes bacterial DNA are packaged by mistake (lytic cycle) transducing particles = viral particles carrying bacterial DNA transfer DNA from previous bacterial host to the new host (recipient) recombination replaces the homologous DNA in recipient cell with DNA fragment from donor cell could transfer ANY DNA fragments with size smiliar to viral genome to recipient cell

operator

binding site of a repressor protein to inhibit transcription initiation

activator (a)

binding site of an activator protein to promote transcription initiation

modified ames test

can be modified to test for mutagenicity of chemicals after they are processed in the liver enzymes in liver can change mutagenicity of test drug

enteric bacteriophage lambda

capsid head, noncontractile tail receptor of phage is maltose porin linear double stranded dna genome encoding ~70 genes

composite transposon (Tn)

carry functional genes from two IS elements (antibiotic resistance genes) whole transposon moves as one unit

enhance competence in gram-negative bacteria

cells in early log phase are used to make competent cells CaCl2 and low temp (4 C) are used electroporation (pulse of high voltage electricity) or heat shock (42 C) drive entry of DNA into cells

CRISPR

clustered regularly interspaced short palindromic repeats - arrays of short repeats separated by short "spacer" sequences small RNA guided DNA degradation based on sequence complementarity bacteria captures piece of invading DNA and keeps it in crispr cluster progeny cells use it to guide defense against future attacks from same parasitic genetic elements

CRISPR

clustered regularly interspaced short palindromic repeats - arrays of short repeats separated by short spacer sequences small RNA guided DNA degradation based on sequence complementarity bacterium/archaea that manages to survive phage attack captures piece of invader's genome and keeps it in CRISPR cluster, uses to guide defense against future attacks from the same parasitic genetic elements bacterial adaptive immune system to invading DNA do not enocde proteins but work as noncoding regulatory RNAs

gene expression

constitutively expressed genes (continuously expressed) - housekeeping genes inducible genes = highly expressed only when needed - in the presence of an inducer (usually have a low, basal level of transcription) repressible gene - transcription level decreases in the presence of a corepressor

beta-galactosidase (LacZ)

converts lactose to glucose when at low levels, converts lactose to allolactose, which binds to Lacl proteins, reducing its affinity to the operator

core vs flexible genome

core = necessary housekeeping genes ie rRNAs, tRNAs, vertically transferred, conserved functions flexible genome - niche specific and variable ie plasmids, genomic islands, transposons, horizontally transferred, niche specific functions

generalized transduction

could transfer any bacterial dna fragment with a size similar to the viral genome into the recipient cell 1. P22 phage DNA infects a host cell and makes subunit components for more phage 2. DNA is packaged into capsid heads, some capsid package host DNA 3. new phage assembly is completed 4. cell lyses, phage is released 5. transducing phage particle injects host dna into new cell, where it may recombine into the chromosome

Anti-CRISPR

counterdefense by phages most common mechanisms - mutations in the crRNA - targeting sequence bacteriophages encode a variety of CRISPR inhibitors

restriction endonucleases

degrades foreign DNA in recipient cells - endogenous DNA protected by modification cut at specific restriction sites - palindromic sequences

interrupted mating experiment

determine frequency of gene transfer from Hfr to F- cells interrupt mating at diff time points transfer frequencies of specific genes in F- cells are determined, genes more distant from oriT have lower transfer frequencies Conjugation bridge (sex pilus) is fragile, some genes will never be transferred

Ames test

determines whether a chemical is a mutagen by detecting DNA mutations in the presence of said chemical uses bacterial auxotrophic mutant - mutant that can't synthesize histidine and therefore is unable to grow on media lacking histidine mutagens cause reversion of mutations of the mutated hisG gene (mutated back to wild type - revertants which regain the ability to grow on media without histidine)

T4 phage gene regulation

does not encode core RNA polymerase early genes > transcribed by E. coli primary sigma factor (70). effectively compete with bacterial housekeeping genes, encode restriction/modification enzymes (protect viral DNA, cleave bacterial DNA) and transcription regulators middle genes: transcribed by E. coli primary sigma factor and viral transcription activator proteins. encodes DNA metabolism proteins (helicase, primase, ligase) transcription regulators. viral DNA replication Late genes: transcribed by viral sigma factor 55 and activator proteins. produce viral structural proteins and cell lysis enzymes. encode: capsit/tail proteins, lysozyme (degrades cell wall), holin (damage cell membrane)

circularly permuted

each phage head contains the same genetic information with different regions at ends

trp operon

encodes enzymes responsible for tryptophan biosynthesis when tryptophan is plentiful, expression of trp operon is repressed at transcription initiation stage by TrpR TrpL (leader) contains the attenuator sequence: transcribed and translated before structural genes 1. binding of Trp to the aporepressor makes the holorepressor (TrpR + tryptophan) 2. binding of holorepressor to the operator inhibits but does not altogether prevent transcription

phage lambda genome

ends of linear phage lambda genome each possess a cos site (a sequence of 200 bp that was generated through cleavage by a terminase enzyme after viral DNA replication) after phage DNA is injected into bacterial cell, the cos "sticky ends" anneal and phage lambda forms a circular genome immediate early genes: "control proteins" - lysis/lysogeny switch early genes - transcribed from either promoter Pl (left operon = enzymes for lysogeny: integrase, excisionase) or Pr (right operon - viral dna replication enzymes) late genes: virion parts, cell lysis enzymes, terminase - all transcribed as one operon from promoter Pr

non-homologous end joining

error prone dna repair triggered by double stranded DNA breaks multitasking enzyme: LigD, acts as a polymerase, exonuclease and ligas introducing errors, often adds/deletes a few nucleotides (frameshift mutation)

regulatory sequences of DNA

exhibits sequence symmetry and contains "inverted repeats"

crRNA processing

expression CRISPR array is transcribed as one precursor RNA and then processed to generate crRNAs each crRNA contains one spacer and one repeat repeat sequence forms specific secondary structure

catabolite repression

expression of genes to metabolize other sugars are repressed in the presence of a more favorable nutrient (glucose)

dynamic bacterial genome

extensive gene loss and gain through mutations of single bases during replication (slow, accumulates through time), large deletions and insertions of large DNA fragments (inter- and intra-species gene transfer by plasmids) natural selection - transferred DNA confer growth advantage

regulator

gene encodes a regulatory protein, which controls gene expression after binding to an operator or activator ligands associating with the regulator protein - inducer or corepressor

regulation of gene expression

gene regulation = 3 levels 1. post translational control = no enzymatic activity 2. translational control - no protein synthesis 3. transcriptional control = no mRNA synthesis

transformation

gene transfer 1.bacteria secretes competence factors 2. accumulation of CF induces the assembly of transformasome located in the cell membrane (quorum sensing) 3. transformasome binds extracellular DNA for uptake

diauxic growth

glucose is metabolized first uptake of other sugars = prohibited expression of genes to metabolize other sugars is induced only after glucose is used up lag phase due to switch in gene expression: biphasic growth curve

transformasome

gram positive bacterial cell membrane protein complex that binds extracellular DNA for uptake

headful packaging

individual genome are cut at positions slightly more (105%) than one complete set of T4 genes

mutagens

induces mutations (10^-3 - 10^-5) physical: x rays and UV chemical: carcinogens, compounds interfere with DNA chemistry, leads to incorrect base pairing. biological: transposons inhibit DNA replication and transcription large scale chromosomal changes

replication of T4 DNA

injects linear DNA genome into E. coli linear DNAs would be progressively shortened if they were replicated through bidirectional replication T4 genome contains repeats at the ends - terminal redundant ends (TR)

genomic islands

insertion of a large segment of DNA from another species into a bacterial genome (chromosome or plasmid) introduces novel functions ie pathogenicity, antibiotic resistance HGT process introduces "direct repeat" sequences at the borders insertion usually occurs at highly conserved genomic regions ie tRNA allowing interspecies transfer

bacteriophage T4

large compact DNA genome ~300 genes enzymes: cell lysis, DNA replication, nucleases, DNA modifying enzymes structural proteins transcription regulators life cycle from entering E. coli to its destruction takes approx 25 minutes 1. Dna injection > formation of early mRNA. DNA of host is degraded 2. phage dna replicated > late RNA made 3. head and tail made >> head filled >> virions formed 4. host cell lysis

lambda phage

lysogenic, enters lytic cycle when induced or low MOI linear DNA genome with cohesive ends (cos sites) Circularizes via the cos sites, omega replication and then concatemers formed through rolling circle replication packaging = terminase cuts at cos sites

T4 phage

lytic life cycle linear DNA genome with terminal redundant ends (TR) bidirectional DNA replication then forms concatemers through homologous recombination headful packaging (105% of genome size)

low tryptophan level (not very low)

majority of ribosomes do not stall at the two trp codons, but reach the translation stop codon of the trpL mRNA 3-4"

lysogeny pathway

makes CII and CI 1. multiple phages infect: CII level high 2 multiple phages infect: CIII level high 3. CII induces CI synthesis from PRE 4. CIII prevents degradation of CII by HflB protease block lytic cycle CI activates PrM to make CI and blocks all lytic cycle transcription from PL and PR.

bacterial defense

modification/mutation of receptors - prevents phage attachment restriction endonucleases cleave injected viral DNA CRISPR - small RNA guided DNA degradation based on sequence similarity

mutations

mutant - organism that is direct offspring of "normal" member of species (wild) but is diff in DNA sequences genetic variation heritable changes in DNA/RNA sequences in the genome introduces novel functions

small regulatory RNAs

noncoding RNAs that regulate the stability and/or translation of target mRNAs regulation is economical/rapid because does not involve protein production post transcriptional control can promote translation

incoming phage DNA survives

plasmids exist in the recipient bacteria as extrachromosomal DNA could incorporate into recipient bacterial genome by homologous recombination - replacing endogenous DNA

pilus

protein filament anchored to bacterial cell surface, contractile genes encoding the pilus are locating on the fertility plasmid (F factor) which is self transmissible (DNA transfer coupled with rolling circle replication)

multiplicity of infection (MOI)

rate of phage particles to host cells

maltose porin

receptor of phage lambda

CRISPR-mediated genome editing

regulates gene expression precisely edits the genome to fix mutations

regulation of E. coli Sigma H

regulation of alternative sigma factors: usually not at the transcription initiation level - mRNA is constitutively produced temperature sensitive regulation of sigma H translation only occurs at high temperature due to the secondary structure of rpoS mRNA high temps melt the secondary structure and allow translation proteolysis removes a small amount at 30 celsius, requires chaperone activity stabilized at higher temp 42C

transcription repression

repressor-corepressor complex binds DNA and represses target gene

site specific recombination

requires integrase encoded by the phage int gene attR and attL sites are generated after lambda DNA is integrated into genome, preventing further lambda DNA integration

starvation (very low tryptophan level)

ribosome stalls at the two adjacent trp codons in trpL mRNA 2-3 loop = anti-attenuator loop RNA polymerase will continue the transcription of trp structural genes >> tryptophan is produced

DsrA

sRNA that activates RpoS translation

targeting invading DNA

secondary structure formed by the repeat sequence recruits Cas proteins, usually nucleases crRNAs guide the cleavage of the invading DNA based on sequence similarity invasing/infection is blocked

insertion sequence (IS elements)

simplest transposable element transposase gene (Tnp) is flanked by inverted repeats (IR) which bind to transposase proteins

excision of lambda prophage

site specific recombination between the attL and attR sites in the e. coli genome excisionase - excises phage DNA together with the integrase from the host genome lambda enters the lytic cycle and starts viral DNA replication

spacer acquisition

small piece is removed from the invading DNA and inserted into CRISPR locus as spacer. molecular recording: spacers are incorporated in sequential order CRISPR locus reflects the history of infection/invasion in the bacterium/archaea

specialized transduction

some viruses integrate its genome into the bacterial chromosome - prophage - which is excised from the bacterial genome before viral DNA replication errors occur during excision process - bacterial DNA adjacent to prophage is mistakenly excised, replicated and packaged into viral capsids transferred bacterial DNA is restricted to the genomics region flanking the prophage in the donor cell

re-enter lytic cycle

stimulated by stress (UV damage, haltered DNA replication, SOS response) RecA is activated as a coprotease and stimulates the self cleavage of CI allows expression of Cro: inhibiting the transcription of CI, phage enters lytic cycle

lambda is a ____ phage

temperate

Avey and Mcleod 1943

tested polysaccharide, protein, RNA and DNA - proved DNA was genetic material that was transferred froms mooth strain to rough strain

guide RNA (gRNA)

the RNA that guides Cas9 to specific sequences in the genome in the most commonly used CRISPR genome engineering system, cleaves double stranded break. nonhomologous end joining introduces errors >> mutagenesis

low concentration of tryptophan

trP is inactive, but transcription is attenuated by TrpL about 15% of transcription can be completed

cis-antisense RNAs (asRNA)

transcribed from sense strand of DNA - base pairing with mRNA transcribed from the template strand to regulate mRNA stability and/or translation function as negative regulators

RNAIII of s. aureus

transcription of RNAIII controlled by quorum sensing activates/represses multiple mRNAS contains multiple stem loops that base pair with different target mRNAs

Lac operon

transcription of the lac ZYA operon only occurs when glucose level is low and lactose is present under both positive and negative controls (Repressor = Lacl - senses presence of lactose, activator = cAMP receptor protein senses low level of glucose) lactose derepresses the lac operon. in the absence of lactose, lacL binds as tetramer to the operator region, represses the lac operon by preventing an open complex formation by RNA polymerase

horizontal gene transfer

transfer of genes from one organism to another (donor to recipient ) allows for fast evolution

gene transfer processes

transformation: a form of horizontal gene transfer that involves free (naked) dna taken up directly from the environment into recipient cell plasmid transformation - used in laboratories transduction: DNA transfer mediated by virus (bacteriophage) conjugation: cell-cell physical contact (sex pilus)

lactose permease (lacY)

transports lactose through cell membrane

transposition

transposase cuts DNA at the inverted repeat sequences on both ends of the transposable element and the target sequence of the target DNA and then ligate the transposable element with the target DNA

trp attenuation

trpL mRNA is being translated before the RNA polymerase reaches the trp structural genes four regions that potentially form 1-2, 2-3 or 3-4 stem loops attenuator loop = 3-4 loop has downstream U-rich region which causes transcription termination two adjacent tryptophan codons within region 1 sense the cellular concentration of tryptophan ribosomes will stall at the trp codons if cellular concentration of tryptophan is very low ribosomes will encounter the stop codon if tryptophan concentration is low (not very low)

starvation

trpR is inactive, attenuation is inactive, high transcription levels of the trp structure genes

Griffith's mortality experiment 1928

two strains of Streptococcus pneumoniae: smooth and rough

phage anti-defense

viral dna modified after replication contains 5-hydroxymethylcytosine, similar to methylation to protect viral DNA from bacterial restriction enzymes anti-crispr ie changes in targeted sequence

lytic cycle

viral infection of bacteria 1. phage attaches to host cell/inserts DNA 2. linear dsDNA cyclizes to circular DNA 3. viral DNase cleaves host cell DNA. Cell synthesizes capsid proteins 4. cell replication phage DNA, DNA is packaged into capsids 5. phage lyses cell and progeny phases are released

lysogenic cycle

viral infection of bacteria 1. phage DNA integrates into host genome to form prophage 2.integrated phage DNA reproduces with host genome 3. integrated phage DNA replicates with host genome 4. stress induces excision of phage DNA 5. phage recombines by re-joining the ends of its phosphodiester chain and enters lytic cycle