Lecture 16 Flashcards
constitutive
genes that are expressed at all times
Not all genes are constitutive because
Optimal energy efficiency
Gene products or pathways may be incompatible
In multicellular organisms → different cell types express unique gene combinations to achieve specific phenotypes
Some genes must be expressed at the same time to achieve
a given metabolic function
In bacteria, the protein RNA polymerase is a
multi-subunit holoenzyme
binds to DNA to initiate transcription
Regulation occurs through
protein-DNA interactions
RNA polymerase recognizes and binds
specific DNA sequences called promoters
consentual sequences (TATA, CAT) - nessary in promoter to have holoenzyme bind
DNA binding proteins tend to have
regulatory functions
DNA binding proteins regulatory functions binding
- bind DNA via amino acid “domains”
- Binding domains can be classified based on conserved structures called “motifs”
types of DNA binding proteins
1) helix -turn- helix → two helixes separated by flexible turn → can insert into DNA
2) Zinc fingers → two slim domains insert → contain zinc ions
3) Lucine Zipper → two slim motifs bind directly to DNA
DNA-protein interactions are conceptualized as
cis-acting elements & trans-acting factors
cis-acting element
a site on a DNA (RNA) molecule that functions as a binding site for a sequence-specific binding protein
cis-acting means that → protein binding to the site affects only DNA (RNA) on the same molecule
trans-acting factor
a diffusible regulatory protein that binds to a specific cis-acting element
Trans-acting factors (proteins) affect → any DNA molecules that are accessible
soluble proteins → can be expressed far away on genome
which is the trans-acting factor?
which is the cis-acting element?
RNA Pol → trans acting → binds to DNA
consensus sequences → cis acting → on same molecule as gene
Bacteria regulate transcription via
operons
→ multiple genes transcribed from a single promoter
→ Only ONE promoter; Only ONE transcript; but multiple genes
→ single transcript translated into multiple proteins
operon
set of regulatory sites and adjacent structural genes (ex. enzymes)
→ cis elements
structural genes
encode (non-regulatory) proteins
promoter drives production through
producing single mRNA
The lac operon model of gene control has____ genes
3 structural
B-galactosidase
permease
transacetylase
all transcribed from ONE promoter (i.e., single mRNA)
translated into separate proteins that function in lactose metabolism
coordinate induction
the simultaneous synthesis of several proteins stimulated by a specific molecule, the inducer
B-galactosidase (in lac operon)
cleaves lactose (lacZ)
permease (in lac operon)
transports lactose into cell (lacY)
transacetylase (in lac operon)
(lacA)
cis-acting elements comprise promoter region of lac operon
control transcription of structural genes
CAP binding DNA site → binds CAP-cAMP complex
promoter (P) DNA site → binds RNA polymerase
operator (O) DNA site →binds lac repressor encoded by lac i
lac i → repressor
E. coli lac operon is an ____ system
inducible
no lactose present → turned off → not want to waste energy producing lactose
Inducible systems are keyed to
substrates
Inducible systems
tend to ___
repression released only if ____
release of repression allows _____
often encode ______
they tend to have metabolic functions (breakdown of sugars for energy)
under → negative control by a repressor protein
→ if inducer (substrate) present
→ allows transcription
→ encode enzymes that catabolize substrate (not needed if substrate absent)
lac operon is best-studied inducible system
transcription keyed to substrate ____
by _____
for _____
inducer is _____
lactose,
metabolized by lac operon gene products (enzymes)
for energy
inducer is lactose (its derivative, allolactose)
repressor protein has a ____ site
repressor protein also has a different, _____ site
DNA-binding → binds the operator → keeps RAN pol. from transcribing
allosteric site → binds the inducer (allolactose)
inducer → substrate/molecule nessary for releasing repressor (changes represor confotrmation)
Presence of lactose
releases repression
when present, lactose (allolactose an inducer) →bindsrepressor → repressor fallsoff the DNA → allows transcription
repression never completely shuts down transcription of the lac operon. Even with an active repressor bound to the operator
there is a low level of transcription,
Mutations in regulatory DNA sites of operon uncovered _____
Mutations in repressor gene uncovered ___
Uncovered cis-acting operator and promoter (CIS ACTING)
Uncovered trans-acting repressor (encodes lac I)
Mutations in structural genes of operon_____ and ____
uncovered _____
Missense mutations (amino acid replacement) → uncovered individual protein functions
Nonsense mutations (stop codon) → POLAR → eliminated multiple proteins; revealed polycistronic mRNA
nonsense mutations affect
translation, not transcription
called polar → presence in one gene blocks translation of that gene and all downstream genes
BECAUSE → Normally → stop and then another start codon very close to each other → ribosome just “slides over”
nonsense mutation → stop codon → stops but far away from another start → ribosome disassociates from mRNA → no lac Y or A
polycistronic mRNA
a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs
Other principles uncovered by analysis of mutations in partial diploids
F factor is an episome
able to replicate as a circular plasmid or integrate into the bacterial chromosome
Integration sites are variable
→ Hfr strains can have F in different positions
→ integrated F factor can excise and re-form as plasmid
Episome
special type of plasmid, which remains as a part of the eukaryotic genome without integration.
F factors can insert or excise from the bacterial chromosome by a → _____
Some bacterial chromosome can be removed during ____ can be _____
causing ____
single crossover event
excision → can be imprecise
Abnormal outlooping → creates F’ factors with bacterial chromosomal DNA
E.g., F’(lac) → can take some of lac operon w it
F’(lac) factors (with lac mutations) can be transferred into E. coli cells (are WT) creating _____
partal diploid → merozygote → for lac
merozygote
A bacterial cell having a second copy of a particular chromosomal region in the form of an exogenote.
Partial diploid analysis shows repressor is
trans-acting
WT lac operon → makes repressor → is transfusable/trans/can move → binds to operator in both WT and mutant transcripts
→ WT rescues → making mutant “normal”
Partial diploid analysis shows the repressor contains a ___ site
super mutant repressor mutation → version of repressor____
lactose-binding allosteric site
super mutant repressor mutation → version of repressor w/mutation at allosteric site → cannot bind to lactose (inducer) → lactose binds but is never released
→Transcription is permanently blocked from both DNA molecules, regardless of whether lactose is present
Partial diploid analysis shows that operators are ____
cis-acting
O+ cannot rescue Oc (they are cis-acting DNA sites)
in WT → represor binds → expression blocked
in mutant → repressor cannot bind to altered operator → cannot block RNA pol from binding → always expresson of structural gene → always transcription
If lactose and glucose are both present, cell prefers
glucose
glucose must be used up first →
only under low glucose is cyclic AMP formed
cAMP must complex with catabolite activator (CAP) to promote transcription of lac genes
cAMP must complex with ____ to promote transcription of lac genes
catabolite activator (CAP)
High Glucose / High Lactose
High Glucose / Low Lactose
Low Glucose / Low Lactose
Low Glucose / High Lactose
Low Glucose / High Lactose
Low Glucose / Low Lactose
High Glucose / High Lactose
High Glucose / Low Lactose
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I+P+O+Z+ / I+P+O+Z+
+ Lactose → Yes
- Lactose → No
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I-P+OcZ+ / I+P+O+Z-
+ Lactose → Yes
- Lactose → Yes
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I+P-OcZ- / I-P+OcZ+
+ Lactose → Yes
- Lactose → Yes
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
IsP+O+Z+ / I+P+O+Z-
+ Lactose → No
- Lactose → No
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
IsP+O+Z+ / I-P+O+Z+
+ Lactose → No
- Lactose → No
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I-P+OcZ+ / I-P+O+Z-
+ Lactose → Yes
- Lactose → Yes
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I-P-O+Z+ / I-P-OcZ+
+ Lactose → No
- Lactose → No
Look at P → if - → no promoter → always no
glucose is ABSENT
will active B-galactosidase protein be produced in the presence or absence of lactose?
I+P+O+Z- / I-P+O+Z+
+ Lactose → Yes
- Lactose → No
In which genotype(s) is B-galactosidase:
Constitutively expressed?
Regulated?
Uninducible?
Constitutively expressed → always expressed → 2, 3, 6
Regulated → normal so + lactose yes - lactose no → 1,8
Uninducible → never expressed → 4,5,7
