4.4.19 Bacterial Genetics I Flashcards
Define genetics, genotype, phenotype, & gene
- Genetics - study of organisms at the level of genes & their products (facilitated by manipulation of genetic material)
- Genotype - genetic makeup of an organism at the nucleic level
- Phenotype - observable trait of the genotype
- Gene - region of DNA (or RNA) that encodes for protein or functional RNA
Define open reading frame, allele, & locus
- Open reading frame (ORF) - a region of the gene that contains the actual genetic information for polypeptide (translated region)
- Allele - different forms of a gene
- Locus - a region in the genome of an organism
Define epigentics & strain
- Epigenetics - study of heritable phenotypic changes that are not based on genetic alterations (mutations) but from differential gene expression due to modification of bases (C or A are most common) or proteins
- Strain - a subdivision of species
Domain > phylum > class > order > family > genus > species > strains
Define mutation, wildtype, mutant, parent, isogenic
- Mutation - inheritable changes in DNA/RNA sequence
- Wildtype - refers to either a gene or strain isolated without “known” mutations
- Mutant - gene or organism w/ mutations
- Parent - refers to either a gene or a strain that served as the predecessor for a given mutant
- Isogenic - all derived from the same parent
What’s the central dogma & reverse transcriptate?
Central dogma: DNA → RNA → protein
Reverse transcriptase: DNA made from RNA template
Steps for DNA replication in bacteria
- Formation of prepriming complex to open up oriC
- Binding of SSB to prevent zipping back & to protect ssDNA
- Formation of replication forks & breaking of H bonds by helicase
- Gyrase relaxes positive supercoiling front of the fork
- Synthesis of short RNA primer by primase (RNAP specializing in DNA replication)
- Bidirectional chain elongation
- Topo IV reintroduces negative supercoiling on new DNA
- Removal of RNA from Okazaki fragments by DNAP I w/ 5’-3’ exonuclease activity & 5’3 DNAP activity
- Joining of the fragments by DNA ligase
- Separation of chromosomes
Most bacterial have ___ chromosomes
circular
DNA replication is ___
Semiconservative
Leading strand vs. lagging strand
- Leading strand - DNAP synthesizes the new strand in 5’ to 3’ direction as 1 long strand
- Lagging strand - orientation of template’s opposite to the direction of DNA synthesis by DNAP so the daughter strand is synthesized in short fragments separated by RNA primers
Describe initiation in transcription
binding of RNAP & transcription factors to promoter
- Formation of closed complex: binding of RNAP to promoter region
- Formation of open complex: unwinding of DNA at -10 region
- Transcription start: at +1 region
Describe elongation in transcription
- Begins after formation of first phosphodiester bond
- Sigma factor lost
Describe termination in transcription
- Cessation of elongation
- Release of transcript from ternary complex
- Dissociation of RNAP from template
- Zipping back of DNA double strands
Factor dependent vs. factor independent
Factor independent
- Hairpin loop rich in G & C followed by string of U’s that bind to A’s (weak bond causes dissociation)
–Combination of 2 RNAP pausing sites
- DNA template rewinds to form double helix
Factor dependent
- Rho uses ATP to break apart H bonds between DNA & RNA to separate transcript from template
Define wobble base. Why is the genetic code “degenerate”?
Wobble base - third position of codon
- A tRNA that recognizes a Pu at the 3rd position of a codon can recognize the other Pu & vice versa w/ Py
What are the “nonsense” codons?
UAA, UGA, or UAG
Don’t code for an AA
Define tRNA
set of adaptor molecules that brings specific AA into translation machinery via anticodon:codon interactions
- Reads codon & puts specific AA into protein at codon site
- AA is at 3’ end
Describe initiation in translation
- requires IFs & charged initiator tRNA
- Position of ribosomes on mRNA
- Selection of proper “reading frame”
- Determined by shine-dalgarno sequence
Describe elongation in translation
- Aminoacyl-tRNA enters A site and pairs with codon
- Peptide bond forms between AA (at the carboxyl group) on P site and AA in A site
- Translocation of growing peptide chain onto P site while emptying out the A site
- A, P, & E sites ratchet back & forth
Describe termination in translation
- Activation of peptidyl transferase hydrolyzes the bond between the polypeptide/tRNA at the P site & results in release of polypeptide
- Dissociation of 70S ribosome by RRF & EF-G:GTP
Describe the A, P & E site
A = acceptor site = aminoacyl tRNA binding site & accepts charged tRNAs
P = peptidyl transferase site & contains previous tRNA attached to nascent polypeptide
Where polypeptide grows
E = exit channel for uncharged tRNA
Ribosomes positioned to correct reading frame by binding to ribosome binding site (shine-dalgarno sequence) via ___
H bonding
Define polysomes & purpose of coupled transcription & translation
Polysomes - complex of several ribosomes attached to mRNA
Purpose:
Increase rate of protein synthesis
Protect mRNA from nucleases
What is the core & holo made up of in RNAP?
Core: (alpha)(2)(beta)(beta’)(omega)
Holo: core + sigma
Define sigma
- promoter recognition for specificity of RNAP
- Tells it where to start transcribing based on promoter location
- Controls gene expression of a given set of genes
Define promoter
- site for binding of RNAP for initiation recognized by sigma
- Located on -35 or -10 region
Closer the sequence of the promoter is to the consensus sequence = ___
Genes w/ stronger promoters are ___
Stronger promoter
frequently transcribed
Define single gene, regulon & transitionally coupled
- Single gene - single transcript & single translation start
- Regulon - Multiples genes are regulated together but physically separated
- Translationally coupled - transcription & translation of the downstream gene’s dependent on the transcription/translation of the upstream gene
Define polarity, transcriptional polarity, & translational polarity
- Polarity - expression of downstream gene affected by a mutation of the upstream gene
- Transcriptional polarity - insertion of a DNA sequence that disrupts transcription
- Translational polarity - mutation that results in creation of a non-sense codon in the upstream gene causes translational polarity on the translationally cooped downstream gene
Define point mutations & leaky mutations
Point mutations
- Can result from mistakes in replication, recombination, or DNA repair
- Incorporation of a wrong base during replication is normally due to tautomeric shift of bases (bases change in base binding)
Leaky mutations
- AA change only slightly affects function
Define tautomer
structural isomer of each base due to spontaneous rearrangement of bonds
Define mutagens & what does radiation cause?
Mutagens - agents that cause mutations
Radiation causes Py dimers (Distorts the double helix, disrupts H bonds, & impairs DNAP)
Define recombination & its purpose
- Recombination - breaking & rejoining of DNA molecules in new combination
Purpose:
- DNA repair
- Introduction of genetic variation for adaptation/evolution
- Genetic rearrangements
Are deletions of more than a few nucleotides are very stable? Are duplications & inversions? Why or why not?
yes, b/c they don’t revert under most circumstances
no, duplications are unstable & revert at a high frequency (bacteria don’t like)
no, inversions are unstable & revert at a high frequency
What affects the stability of a mutation?
What is mutated & how does the mutation affect the physiology of the organism
What type of mutation
Define DNA proofreading/editing
- senses mutations due to topological constraints
- Uses 3’-5’ exonuclease to chew back & remove wrong bases & uses 5’-3’ polymerase activity to synthesize the strand
Define mismatch repair
when proofreading isn’t enough
mismatch repair proteins bind & catalyze removal of a segment of a newly synthesized DNA including the mismatch
What’s the error rate of DNAP?
10^-5 to 10^-6
Define nucleotide excision repair
distortion in helix recognized & removed by exonuclease
specific endonucleases cleave the strand w/ mutations
gap repaired by DNAP
ligase repairs the break in phosphodiester bond
Define base excision repair
removal of altered base by specific N-glycosylase
AP endonuclease removes sugar & P
Describe photoreactive repair & define photolyase
Photolyase - gets energy from light
- Uses FADH2 as a cofactor
- Scans DNA until Py dimer
Visible light in 350-500 nm region absorbs light & facilitates removal of T-T dimer & allows replication to continue
Define end-joining repair
broken ends are processed by nuclease & end-joined together
Define mobile genetic elements, transposition & transposase
- Mobile genetic elements - genetic elements that can “move” or “hop” around
- Transposition - movement of DNA mediated by transposable elements (transposons)
- Transposase - enzyme catalyzing transposition
Define insertion sequences
small clusters of genes (usually 1 or 2) that encode for the movement of a DNA fragment
- The DNA fragments are surrounded by inverted repeats at each end w/ transposase in middle
Define composite transposons & noncomposite transposons
Composite transposons - 2 insertion sequences w/ genes in the middle
- Only 1 of the insertion sequences encodes active transposase
Noncomposite transposons - inverted repeat, transposon, & gene of interest that you can carry & follow movement
What are the 2 different mechanisms of transposition in bacteria
- Cut-&-Paste - transposon removed from donor DNA
- Replicative transposition - 2 copies of transposon can serve as a region of homologous DNA that can recombine & move 1 segment into another
Describe plasmids. Why study them?
- autonomously replicate from the chromosome & produce progeny
- Divide equally among daughter cells
- They’re how we clone things
- Encodes antibiotic resistance (are transferable from 1 strain to another)
—Allows antibiotic genes to move through various populations - Adaptation & evolution
Define bacteriophages, lysogeny phage, & lytic phage
- Bacteriophages - bacterial viruses that live off bacteria
- Lysogeny phage - have ability to infect cell & integrate into host genome
- Lytic phage - have ability to infect host cell, replicate & kill host
Describe the 3 genes in temporal regulation of gene expression in phage
Early genes: expressed by host enzymes
Middle genes: require host & phage enzymes for expression
- May repress early genes or induce late genes
Late genes: require host & phage middle gene products
- Mostly phage
- Mostly for capsid formation & phage release from cells
Within the lysogeny phage, there’s prophage, lysogen & lysogenic conversion. Define these 3
- Prophage = phage that’s silent & maintained in host
- Lysogen = bacterial cell w/ a prophage
- Lysogenic conversion = phage causes a change in phenotype; may convert nonpathogenic bacterium into a pathogen by a phage
