Dunn M&C - Prokaryotic Transcriptional Regulation

promoter

initiation of transcription; directs the transcription machinery to the 5’ end of a gene

terminator

termination of transcription; causes RNA polymerase to dissociate from the template DNA, thereby ending transcription

transcription factor

sigma factor — e.coli that is required for transcription

transcription three stages

1. initiation

2. elongation

3. termination

applies to both prokaryotes and eukaryotes

regulatory transcription factors

determine how efficiently (and therefore frequently) a transcriptional initiation complex forms and successfully escapes the promoter

positive regulation

leads to transcriptional activators

transcriptional activators

protein that promotes transcription; helps recruit RNA polymerase or make initiation more efficient

negative regulation

leads to transcriptional repressors

transcriptional repressor

protein that inhibits transcription; prevents recruitment of RNA polymerase

genes

can have intrinsic differences in sequence that cause them to be expressed with different efficiencies

gene expression

can be regulated at any step from transcription to protein activity

• transcriptional regulation is most common way of regulating genes, in part because it saves the cell the most energy/resources

• often controlled by the presence of small molecules that provide info about the environment, often nutrient availability

• the small molecules control transcription by allosteric interaction w/ factors

transcription factor

a protein, usually with DNA-binding activity, that influences the rate of transcription of a gene, either positively or negatively

• have to find their binding sites in a vast genome of sequence

p53 protein

Anti-cancer proteins that we have, prevent tumor formulation

gene regulation

simpler than in eukaryotes because:

• DNA is not packaged into chromatin

• prokaryotes streamline their processes for efficiency (to minimize energy expenditure

microbiome

all of the microorganisms that live on your skin, in/on your mucosa (ear, mouth, nose), and in your gut

commensal bacteria

“helpful bacteria” that assist with digestion and protect against opportunistic pathogens

prokaryotes and transcriptional regulation

metabolic machines, gene expression is often controlled by the presence of small molecules

• regulatory transcription factors

• direct regulation by nutrients & small molecules

• existence of operons

• limited combinatorial control

eukaryotes and transcriptional regulation

• regulatory transcription factors

• regulation by covalent modification of chromatin & its folding state

• lots of combinatorial control

lac operon

illustrates prokaryotic transcription regulation

• contains gene coding sequences

• lacZ

• lacY

• lacl

operon

a region of DNA that includes the coding sequence for multiple genes that get transcribed together into a single mRNA (polycistronic mRNA).

• common in prokaryotic, very rare in eukaryotes

polycistronic

single molecule of messenger RNA (mRNA) that contains the coding sequences for two or more different proteins

lacl gene

located near the operon and encodes the lac repressor protein

• expressed constitutively at a low level

• in the absence of lactose, the repressor binds the operator, and consequently RNA pol is not recruited to the promoter

lacZ

the gene for beta-galactosidase, which cleaves the lactose molecule into its glucose and galactose constitutents

lacY

the gene for lactose permease, which transports lactose from the external medium into the cell

regulatory sequences

• promoter

• operator — the binding site for a repressor protein

• CRP-cAMP binding site —binding site for a positive regulator

lacI gene repressor

in the presence of lactose, the repressor is allosterically inactivated by binding to allolactose

• a conformational change inactivates this through allosteric regulation

allolactose

a lactose metabolite

lacO gene operator

in the presence of lactose, it cannot bind to the operator and transcription is permitted. thus proteins that are needed to use lactose as a carbon source (energy source) are produced.

CRP-cAMP complex

a positive regulator of the lac operon that controls transcription based on glucose availability

• low glucose → high cAMP

• high glucose → low cAMP

provides an additional level of control over transcription that is sensitive to glucose levels

• because carbon/energy courses are preferable to others, so if given high glucose and lactose, the cell will use glucose, but if glucose is depleted, it can invest in lactose utilization

lacZ and lacY

loss of a function mutation in the sequence encoding the lac repressor or in the operator sequence causes LacZ and LacY to be constitutively expressed

constitutively

under all conditions - regardless of the presence/absence of lactose and glucose

trp operon

encodes genes required for the synthesis of tryptophan, not its utilization; thus, tryptophan makes the repressor bind rather than fall off the DNA

• when tryptophan is present, the trp repressor binds the operator, and RNA synthesis is blocked.

• when tryptophan is absent, the repressor dissociates from the operation and RNA synthesis proceeds

high tryptophan

repression

low tryptophan

de-repression

amino acid bonds

peptide (covalent) bonds