week 6/7 ish Flashcards
what does it mean that microbial chromosomes are streamlined
less intergenic space, smaller regulatory regions than larger eukaryotic counterparts
operons
clusters of genes that share the same promoter and are transcribed as a single large mRNA
bacterial transcriptional unit
true or false: introns are very rare in yeast and prokaryotes
true
intergenic sequences
promoters, enhancers, repressors, spacers
true or false: intergenic sequences are bigger in prokaryotes
false, smaller
microbes are extremely “gene……” compared to multicellular eukaryotes
dense
true or false: the majority of eukaryotic DNA is the essential, backbone genetic information
false; non-coding dna accounts for a greater percentage
operons consist of 4 main parts:
upstream activator site
promoter
operator
structural genes
polycistronic
contain multiple ORF that encode amino acids
upstream activator site
DNA sequence upstream of a promoter that increases transcription via binding of activator proteins
promoter
region on DNA to which RNA polymerase binds to initiate transcription
specific to sigma factors
sigma factors
interchangeable subunits of the RNA polymerase
operator
DNA sequence which decreases transcription via binding of repressor proteins
usually overlaps promoter
structural genes
genes encoded by the operon
why would genes be found on the same operon
belong to the same pathway
account for different subunits of a particular protein
gene expression is highest for genes at the……end of the transcript
5’
polar effects
disrupt translation of downstream genes
4 examples of operons hosting genes with related functions
arg
lac
trp
fim
gene expression is highly…….
coordinated
(regulons)
ribosomal release factors and signaling sequences are needed to ensure proper…..
translation
unlike eukaryotes, bacteria do not have a membrane-bound…….so mRNAs are not……..or……or……before translation
nucleus
capped, tailed, targeted
in prokaryotes, translation can begin before
transcription is completed
beads on a string
multiple ribosomes can load onto the nascent transcript
poly-A tails are signals of……in prokaryotes
decay
transcription in bacteria begins with:
RNA polymerase fusing with the sigma factor
Sigma Factor
promoter-recognizing subunit
the Sigma Factor recognizes the…… that is located…..
Pribnow box
upstream from the mRNA start site
(-10 and -35 regions)
sigma factors allow for a coordinated……response
transcriptional
o70 RpoD
E. coli sigma factor sequence and function
TTGACA
normal growth
o38 RpoS
CCGGCG
general stress response
regulon
sigma factors
or
activators/repressors
facilitate coordinated expression of genes and rapid response to environmental stimuli
once transcription begins, the sigma factor…..
releases
transcription then continues until……
the termination site is reached
transcription termination in bacteria:
one goal, two possibilities
possibility 1 termination:
factor independent termination: stem-loop followed by a U poly-track
possibility 2 termination:
factor dependent termination: rut site + Rho factor
when transcription is terminated by Rho, the DNA will….
encode a rut site and a Rho-sensitive binding site downstream of the coding region
once transcribed, Rho can bind to the rut site on the nascent mRNA transcript and move up the mRNA until it comes in contact with…….
the paused RNA polymerase
when Rho catches up to the RNA polymerase, it will perform a……..to release the RNA-DNA hybrid. this causes the RNA polymerase to…..
helicase activity
dissociate and terminate transcription
translation in bacteria uses the….. ribosome
80S
translation initiation is a…..step process
2
step 1 of translation initiation
30S subunit lines up with the ribosomal binding site, initiator tRNA binds
how is the start codon ensured to be aligned in the P-site
16S ribosomal RNA aligns with the Shine-Dalgarno Sequence
step 2 of translation initiation:
50S is added and elongation can begin
why/how beads on a string ?
translation can re-initiate as soon as the first ribosome clears the beginning of the transcript
translational coupling
closer together cistrons
@ stop codon, 50S disassociates
30S keeps going, hits next SD and then 50S reassociates and translation restarts
de novo internal translation initiation
further apart cistrons
@ stop codon, entire ribosome disassociates
at next SD, 30S reassociates, 50S binds, translation re-initiates
constitutively expressed
“always on”
inducible
these genes can be “turned on” and “turned off”
what kinds of genes are constitutively expressed
essential genes/ central processes
ex) replication, transcription, translation, main metabolism, etc.
what kinds of genes are inducible
non-essential genes/ auxiliary processes
ex) amino acid, nucleic acid biosynthesis, transporters, etc.
arg operon
encodes proteins involved with the biosynthesis of arginine
“on” by default and can be turned off by repression
when the cell has enough free arginine, then there will be free arginine that can be…..
this causes a conformational change, allowing the ArgR repressor to……
bound by the ArgR repressor
bind the arg operator and stop transcription
arg operon regulation is what type of regulation? and what does this mean
pre-transcriptional
gene product expression is controlled before transcript is made
change in transcription factor’s ability to bind is a form of…….
allosterism
lac operon
encodes proteins involved with lactose metabolism
“off” by default, can be turned on by induction
when the cell encounters and uptakes allolactose, it is bound by the…..
this causes a conformational change, releasing the LacI repressor and inducing……
LacI repressor
transcription
polar effects
operon is not repressed but an early gene gets a nonsense mutation
*genes downstream of the polar mutation have reduced or no expression
fim operon: OFF phase
FimB and FimE are expressed
fim promoter faces the inverse direction
phase variation
a reversible switch between on/off expressing phase
fim operon: ON phase
only FimB is expressed
fim promoter faces the forward direction
post-transcriptional regulation: Attenuation and the trp operon
trp operon has a leader peptide which encodes 2 complimentary stem loops: regular + termination signal with U poly-track
when cell has enough tryptophan, there will be lots of……
ribosome will translate……..and a ……..loop will form
trp-tRNA’s
leader peptide
termination stem
when cell is low on tryptophan, there will be a lack of……
ribosome will stall on…… and an……..loop will form between regions…..and……
trp-tRNA’s
leader peptide
anti-terminator stem
2 and 3
what are mutations
any heritable change in the DNA sequence of a cell
where do mutations come from
mistakes during cell division
exposure to DNA-damaging agents in environment
5 major outcomes of mutations
increase
decrease
change
eliminate
no effect
3 major classes of mutations
base substitutions
indels
structural variants
base substitutions + 3 types
most common mutation
silent–> same amino acids produced
missense–> different amino acid produced
nonsense–> early stop codon
outcome of base substitution is increased function only if……..
the cell needs higher expression of the gene/operon
indels are common but generally restricted to…….regions
repetitive
indel mutations
insertion/ deletion
frameshift - extensive missense
effect of +/- 3 indel
gain or lose amino acid
structural variants are more rare, but effects are…….
larger
structural variants
large deletions/ insertions
gain/loss of gene or operon
6 types of structural variants
deletion insertion
tandem duplication
interspersed duplication
inversion
translocation
2 theories ab mutations/evolution
selection
neutral
selection theory
mutations = deleterious or advantageous
natural selection can act on them all
neutral theory
mutations = rarely advantageous; mostly deleterious or neutral
natural selection cannot act on neutral (no phenotype)
whether a mutation is good/bad/neutral depends on…….
its environment
microbes have the lowest………..but they have huge population sizes which = quick……..
mutation rates
evolution
1.0x 10^6 mutations/mL/generation
1 % mutations are beneficial
= ?
10,000 beneficial mutations per generation
stress-induced mutagenesis
stressful conditions temporarily increase replicative mutation rate
WT mutation rate
higher/steeper curve
blue
hypermutator mutation rate
variability around mode genotype
wider/lower
red
population without mutator cells:
before, during, long term effect
before stress: low genetic variability
during stress: massive cell death
long term: extinction
population of constitutive mutators:
before, during, long term effect
before stress: high genetic variability
during stress: survival of cells carrying mutations
long term: expansion of adaptive mutants
spontaneously/ subpopulations of transiently mutator cells:
before, during, long term effect
before: low overall population mutation rates
during: survival of cells w/adaptive mutations generated in transient mutator subpopulation
long term: expansion of adaptive mutants/ low overall population mutation rates
Ames test
determines of a compound is a mutagen by looking for revertants
- grow His auxotroph in histidine media
- take fraction of culture + expose to possible mutagen
- plate treated+nontreated samples on no-His media
- count colonies after overnight incubation
if possible mutagen contains more colonies than control = mutagen is confirmed
vertical gene transfer
transfer of variation from parent to offspring
horizontal gene transfer
direct transfer of variation between unrelated cells
3 methods for horizontal gene transfer
conjugation, transformation, transduction
conjugation
= discussion of transfer of plasmids
one bacterium transfers genetic material to another through direct contact
transformation
exogenous genetic material is directly taken up and incorporated by a cell through its cell membrane
transduction
DNA is transferred from one bacterium to another by a virus
plasmids are……. acquired and…….. transmitted
horizontally
vertically
low copy number
less than 5 copies
can be larger bc few
medium copy number
5-50 copies
high copy number
50-hundreds copies
can’t maintain large plasmids @ high copy number bc……
it’s energetically draining
replication regulation is related to the plasmid’s……
oriP
1 oriP detected, plasmid can replicate
low copy double plasmids w/ similar trip cannot replicate bc……
2 oriP’s would be detected, can’t replicate; one oriP would be lost in next generation
replication of high copy number plasmids is……..
unregulated
high copy plasmids are partitioned into offspring cells……
randomly
conjugation requires a……….
sex pilus
this sex plus is……..encoded
plasmid
donor cell
contains plasmid encoding pilus
recipient cell:
does not contain plasmid
5 functional types of plasmids
fertility F
resistance R
Col
Degrative
Virulence
F plasmid function
- donor cell attaches w/pilus, draws cells together
- cells contact one another
- one strand of plasmid DNA transfers
- recipient synthesizes a complimentary strand to become an F+ cell; donor synthesizes a complementary strand, restoring its complete plasmid
why is conjugation is risky?
expression of the pills results in susceptibility to bacteriophage
R plasmids
for resistance to antimicrobials, heavy metals, acid, stresses
Col
encode proteins that kill surrounding bacteria, colicines
degrative plasmids
for genes that allow bacteria to break down a new substance
virulence
encode genes that make the bacterium a better pathogen
naive cells can acquire new functions from…..of plasmids
HGT
plasmids are expensive to maintain and can be lost if they aren’t………..
actively needed
recombination ensures that function is…….
retained long term
phage (bacteriophage)
viruses that specifically infect bacterial cells
phage receptors
cell surface proteins that are often targeted by phages for cell access
common phage receptors
flagella, pili, iron transporters, LPS
only……..phage can form a prophage and intergrate into the genome
temperate
cryptic prophages
prophages acquire mutations that trap them on the bacterial chromosome
deletion of different prophages from E Coli results in………
reduced stress tolerance (reduced growth)
transformation
free DNA from the environment is incorporated into a recipient cell and brings about a genetic change
competent
able to take up DNA and be transformed
genetically determined
requires the use of genetically encoded DNA uptake machinery
chemically induced
E. coli must be
requires cells to be treated with chemicals that permeabilize their membranes, allowing DNA to enter the cell
genetically determines competence process
- bacterium lyses and releases its cell contents and DNA
- cell surface proteins on competent recipient cell binds a DNA fragment
- recipient cell uptakes a single strand of the free DNA fragment
- RecA recombination protein facilitates the integration of DNA fragment into the bacterial genome
- competent cell permanently retains new function encoded by the once exogenous DNA
how is E. coli made chemically competent
CaCl2 and heat shock = make membrane permeable
