Bacterial Operons and Eukaryotic Gene Expression Regulation

Three steps of information flow in bacteria

DNA → mRNA → protein → activated protein

Transcriptional control

mRNA is only made for proteins that are needed; controls expression before the cell expends resources (efficient but slow)

Translational control

Not all mRNAs are translated; allows more rapid changes because mRNA is already made (on/off: made into protein or not)

Post-translational control

Proteins must be activated by chemical modification; provides the most rapid response, often via phosphorylation/dephosphorylation

Constitutive expression

Genes transcribed all the time, e.g., housekeeping genes and genes for glycolysis enzymes

Is gene expression all or none?

No; the level of expression can vary between extremes

How does transcriptional control work in bacteria?

Regulatory proteins affect RNA polymerase's ability to bind the promoter and initiate transcription

Ways translational control can occur

Regulatory molecules speed up mRNA degradation; the ability of mRNA to be translated is affected (e.g., an interfering RNA prevents translation)

Why does mRNA persistence matter?

If a message is made and allowed to persist, more protein can be made from it

Negative control

Negatively impacts gene expression (slows protein production); involves a repressor

Positive control

Speeds up protein production; involves an activator (more activator binding gives faster transcription)

Operon

Promoter plus additional sequences that control transcription: the operator and structural genes

Why are genes in an operon turned on/off together?

One mRNA is transcribed that contains multiple genes

Regulator gene

Separate from the operon; DNA sequence encoding products that affect operon function; has its own promoter and is transcribed/translated into a protein that works at the operator

What does the regulatory protein do?

Interacts with the operator and either induces or represses transcription of the operon

Inducible operon

Transcription is normally OFF and needs to be turned on

Repressible operon

Transcription is normally ON and needs to be turned off

Inducible operon: role of the regulator

Binds the operator and keeps the status quo of no transcription

Inducible operon: role of the inducer

Binds the regulator protein, causing a conformational change so it can no longer bind the operator; RNA polymerase can then transcribe

Repressible operon: role of the end product

Serves as a corepressor; binds the repressor, causing a conformational change so it can bind the operator and block transcription

Lac operon function

Involved in metabolism of lactose; if lactose is present, proteins are made to break it down; if not, no energy is wasted

Lac operon type of control

Inducible, negative control

lacZ

Codes for β-galactosidase, which cleaves lactose into galactose and glucose

lacY

Codes for galactoside permease, a pore-like protein that transports lactose into the cell

lacA

Codes for transacetylase; anything left over (sugar) is exported from the cell by this

lacI

Gene for the repressor of the lac operon; always expressed; the protein binds the operator and blocks RNA polymerase

Three central ideas of the lac operon (1)

lacZ, lacY, and lacA are adjacent and transcribed into one mRNA from a single promoter

Three central ideas of the lac operon (2)

lacI is a repressor of the lac operon and is always expressed

Three central ideas of the lac operon (3)

Lactose is the inducer; it binds the repressor and changes the shape of lacI, causing it to release from the operator

Lac operon with lactose present

Lactose acts as inducer; high levels of β-galactosidase are made

Lac operon with no lactose

Everything is turned off

Lac operon when glucose is present

Glucose is used first; bacteria use it preferentially

lacZ⁻ mutant

Cannot cleave lactose because it lacks functional β-galactosidase

lacY⁻ mutant

Does not accumulate lactose in cells because it lacks galactoside permease

lacI⁻ mutant

Produces β-galactosidase and galactoside permease even when lactose is present

Trp operon type of control

Negative control, repressible operon

Trp operon regulator gene

Makes a repressor

Role of tryptophan in the trp operon

Corepressor; allows the repressor to bind the operator, preventing transcription

Trp operon structural genes

5 genes: trpE, trpD, trpC, trpB, trpA

Three differences between eukaryotic and bacterial regulation

Each structural gene has its own promoter and is transcribed separately; DNA must unwind from histone proteins before transcription; transcription and translation are separated in time and space

Three ways chromatin structure affects gene expression

Chromatin remodeling, histone modification (acetyl/methyl groups), DNA methylation

Chromatin-remodeling complexes

Bind directly to DNA sites and reposition nucleosomes

Example of a chromatin-remodeling complex

SWI-SNF

Chromatin remodeling step 1

The chromatin-remodeling complex binds DNA (uses ATP, producing ADP + Pi)

Chromatin remodeling step 2

It repositions nucleosomes, exposing a transcription factor binding site

Chromatin remodeling step 3

Transcription factors and RNA polymerase bind DNA and initiate transcription (mRNA made)

Effect of acetyl groups on histones

Stimulates transcription; disrupts chromatin structure and permits some transcription factors to bind DNA

Histone acetyl groups are added to what part?

The tails of histone proteins

Histone acetyltransferases

"Writers" for acetylation; add acetyl groups

Deacetylases

"Erasers" for acetylation; remove acetyl groups

Effect of methyl groups on histone tails

Activation or repression

Histone methyltransferases

"Writers" for methylation; add methyl groups

Histone demethylases

"Erasers" for methylation; remove methyl groups

Writers, readers, erasers

The three classes of proteins involved in histone modification

DNA methylation

Methylation of cytosine bases adjacent to guanine nucleotides (CpG)

CpG dinucleotide

A cytosine next to a guanine

CpG islands

Clusters of CpG dinucleotides

Where are CpG islands found?

Near the promoters of 50-70% of human genes

Effect of DNA methylation on transcription

Prevents transcription by blocking the promoter regions

Unmethylated cytosines in a CpG island

Activation of gene expression

5-methylcytosine residues in a CpG island

Suppression of gene expression

Transcription factors

Stimulate and stabilize the basal transcription apparatus at the core promoter

Enhancer

DNA sequence that stimulates transcription from a distance away from the promoter

Silencer

DNA sequence with an inhibitory effect on the transcription of distant genes

Insulator

DNA sequence that blocks or insulates the effect of enhancers/silencers

How insulators may function

By causing loops of chromatin that form interacting regions of genes and regulatory elements

Regulatory neighborhoods

Insulators and their binding proteins help create neighborhoods of regulatory elements and genes that can interact but are insulated from elements in other neighborhoods

TAD

Neighborhood of gene regulation; enhancers can interact with promoters within a TAD but not with promoters in other TADs

Proteins at insulators that form TAD boundaries

CTCF and cohesin

When does an insulator block an enhancer?

When the insulator lies between the enhancer and the promoter

Basal transcription apparatus components

General transcription factors and RNA polymerase bound at the core promoter (includes TATA box region)

Other components in enhancer-promoter looping

Transcription factors, mediator, and coactivator

Alternative splicing

Selection of alternative splice sites leads to production of different proteins; a form of regulation through control of mRNA processing

Steps of RNA degradation

5′-cap removal; shortening of the poly(A) tail; degradation of the 5′ UTR, coding sequence, and 3′ UTR

Small RNAs in RNA interference

Small interfering RNAs (siRNAs) and microRNAs (miRNAs)

Dicer

Cleaves and processes double-stranded RNA to produce single-stranded siRNAs or miRNAs

RISC

RNA-induced silencing complex; formed when siRNAs or miRNAs combine with proteins

How RISC finds its target

The RNA component of RISC pairs with complementary base sequences in specific mRNA molecules, often in the 3′ UTR

siRNA vs. miRNA source

siRNAs come from long double-stranded RNA; miRNAs come from a double-stranded region of an RNA

RNA interference outcome 1

RNA cleavage: RISC with an siRNA pairs with mRNA and cleaves it, leading to degradation

RNA interference outcome 2

Inhibition of translation

Importance of mutations

Source of all genetic variation (raw material for evolution); source of many diseases and disorders; useful for probing fundamental biological processes

Somatic mutation

Occurs in nonreproductive cells; passed to new cells through mitosis, creating a clone of mutant cells

Germ-line mutation

Occurs in cells that give rise to gametes; passed via meiosis and sexual reproduction to about half the next generation, who carry it in all their cells

Types of gene mutations by molecular nature

Base substitutions, insertions and deletions, expanding nucleotide repeats

Base substitution

One base replaced by another; alters a single codon

Transition

Substitution of a purine for a purine or a pyrimidine for a pyrimidine (A↔G, C↔T)

Transversion

Substitution of a pyrimidine for a purine or a purine for a pyrimidine (A→C, A→T, G→C, G→T, C→A, C→G, T→A, T→G)

Insertions and deletions

Alter the reading frame and may change many codons; can cause frameshift mutations

Frameshift mutation

Insertion or deletion that shifts the reading frame

Expanding nucleotide repeats

Increase in the number of copies of a set of nucleotides

Fragile-X chromosome

Fragile site on the long arm due to expanding repeats; more than 200 CGG repeats; turns off transcription

Missense mutation

Amino acid changed to a different amino acid (e.g., Ser to Leu)

Nonsense mutation

Sense codon changed to a nonsense (stop) codon; premature termination of translation

Silent mutation

Codon changed to a synonymous codon; no change in amino acid sequence

Forward mutation

Wild type to mutant type

Reverse mutation

Mutant type to wild type

Suppressor mutation

A mutation that hides or suppresses the effect of another mutation

Intragenic suppressor

Occurs in the same gene containing the mutation being suppressed; a second mutation at a different site may restore the original amino acid

Intergenic suppressor

Occurs in a gene other than the one bearing the original mutation it suppresses