Lecture 25 Flashcards
when are tissue/cell/time-specific genes controlled?
controlled at level of transcription
5 unique features of prokaryotic cells
- no nucleus –> transcription + translation occur simultaneously
- 1 circular chromosome
- fewer proteins associated with DNA
- no introns –> no splicing
- genes for 1 metabolic process organized in operon
what is a major point of gene regulation in prokaryotic cells?
transcription initiation
organization of a gene (5)
- promoter
- -35/-10 consensus sequence
- +1 nt = TSS
- ATG (Met)
- gene
what is the role of -35/-10 consensus sequence?
binding site for RNA pol
how is RNA pol recruited to -35/-10 promoter sequences?
sigma factor subunit of RNA pol scans DNA and binds the -35/-10 promoter sequences –> recruits + properly positions the rest of RNA pol at TSS
Describe the default state of prokaryotic gene
ON
sigma factor easily binds promoter since prokaryotic DNA is more simple and not wound in histones
positive regulation
activator binds activator-binding site upstream of promoter to allow RNA pol to bind and allow transcription
what happens when the activator is not bound to the activator-binding site?
there is low transcription
negative regulation
repressor binds operator downstream of promoter to block RNA pol and block transcription
what happens when the repressor is not bound to the operator?
RNA pol can bind promoter so transcription can occur
describe allosteric regulation on activator
effector binds activator –> activator changes conformation so it can bind DNA and activate transcription (gene ON)
no effector –> activator cannot bind DNA or activate transcription (gene OFF)
describe allosteric regulation on repressor
effector binds repressor –> repressor changes conformation so it cannot bind operator and stops repressing (gene ON)
no effector –> repressor binds operator and represses activation (gene OFF)
What is an inducer?
an effector that increases gene expression
what organism is the Lac operon found in?
E. coli
why must the lac operon be regulated?
energetically costly to produce mRNA and proteins –> better to only be active when lactose is available
2 enzymes involved in lac operon
- LacY –> PERMEASE
- LacZ –> beta-GALACTOSIDASE
organization of lac operon
REGULATORY COMPONENTS:
- repressor protein (not in operon)
- promoter
- operator
STRUCTURAL GENES (PROTEINS):
- LacZ
- LacY
3 characteristics of lac operon
- inducible
- under negative control
- under positive control
lac operon when there is NO LACTOSE
repressor protein is produced and binds operator –> RNA pol is blocked –> no transcription
lac operon when there IS LACTOSE
repressor protein is produced but lactose is effector that prevents it from binding operator –> RNA pol is active –> LacZ and LacY transcribed on 1 mRNA
when is I expressed?
I = repressor protein
always expressed!! independent of lactose
synthetic inducer of lac operon
IPTG
3 main classes of mutations
- structural gene mutants
- uninducible mutants
- constitutive mutants
what are structural gene mutations?
affect function of 1 enzyme, the other is inducible
describe the production of beta-galactosidase and permease with Z+Y+ genotype (absence vs presence of lactose)
beta-galactosidase:
- no lactose = no activity
- lactose = activity
permease:
- no lactose = no activity
- lactose = activity
this is WT
describe the production of beta-galactosidase and permease with Z-Y+ genotype (absence vs presence of lactose)
significance of these results?
beta-galactosidase:
- no lactose = no activity
- lactose = no activity (UNINDUCIBLE)
permease:
- no lactose = no activity
- lactose = activity (INDUCIBLE)
since both enzymes are in 1 operon, transcription can occur normally but someting directly affecting beta-galactosidase only
what are uninducible mutants?
can’t make LacZ AND LacY in presence of IPTG –> failure of activation
what are constitutive mutants?
make both LacZ and LacY even in absence of IPTG –> failure of repression
what is a partial diploid?
prokaryote is haploid for everything except diploid for lac operon
2 things that a partial diploid lets you test
- dominant vs recessive
- cis vs trans-acting factors
what is a cis-acting factor?
mutation only affects transcription of genes on same DNA molecule –> mutation in REGULATORY element
what is a trans-acting factor?
mutation also affects transcription of genes on the other DNA molecule –> mutation affects coding of DIFFUSIBLE PROTEIN
what do P mutations affect? how do we know?
haploid E. coli with mutant P makes LacY and LacZ uninducible –> failure of activation
do P mutations act in cis or trans? how do we know?
P mutations affect transcription of genes on the same DNA molecule –> cis
P-Z+Y+ / P+Z-Y- makes LacY and LacZ uninducible, since acting in cis the WT P cannot compensate for P-
what do O mutations affect? how do we know?
haploid E. coli with mutant O makes LacY and LacZ constitutively active –> failure of repression
do O mutations act in cis or trans? how do we know?
O mutations affect transcription on the same DNA molecule –> cis
O+Z+Y+/OcZ-Y- makes LacY and LacZ inducible only, since acting in cis the Oc cannot cause constitutive activity bc Z and Y are mutant
what does it mean when O is constitutive?
repressor never bound so enzymes can be activated
what are the 2 elements of the lac operon that are cis-acting?
promoter and operator
what is the effect of the I mutation?
haploid E. coli with mutant I makes LacY and LacZ constitutively active –> failure of repression
do I mutations act in cis or trans? how do we know?
I mutation affects both DNA molecules –> trans
I+Z-Y- / I-Z+Y+ makes LacY and LacZ inducible, since acting in trans the WT repressor can bind both DNA molecules
what is the Is mutation?
super-repressor mutation –> operon becomes uninducible
what happens if the genotype is IsZ+Y+ / I+Z+Y+?
I+ cannot complement for Is so Is on any DNA molecule makes all systems uninducible
relationship of Is to I+
Is is dominant to I+
how does Is block transcription?
mutation affects allosteric site so inducer cannot bind and repressor stays bound to DNA to block transcription
what allows for positive regulation of the lac operon?
glucose
if glucose and lactose are present, which will E. coli prefer to use?
glucose
how does glucose allow for positive regulation
increased glucose = decreased cAMP
decreased cAMP = CAP can’t bind DNA
CAP can’t bind DNA = no transcription activation
what is the role of CAP
Binds DNA to allow RNA pol to bind promoter
what happens to the lac operon if:
- glucose is present / low cAMP
- no lactose
glucose is present = CAP can’t activate transcription
no lactose = repressor binds operator so no transcription
REPRESSION
what happens to the lac operon if:
- glucose is present / low cAMP
- lactose is present
glucose is present = CAP can’t activate transcription
lactose = prevents repressor from binding operator so there’s some basal transcription
NO REPRESSION
NO ACTIVATION
what happens to the lac operon if:
- no glucose / high cAMP
- lactose is present
no glucose = CAP can activate transcription
lactose = prevents repressor from binding operator
ACTIVATION
