Chapter 28 Regulation of Gene Expression

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基本知識

DNA elements that control transcription

Protein factors that control transcription

Trends in Understanding Gene Regulation

The Vocabulary of Gene Regulation

Ways to Regulate Protein Concentration in a Cell

Synthesis of primary RNA transcript

How to process this RNA into mRNA

Posttranscriptional modifications of mRNA

Degradation of mRNA

Protein synthesis

Posttranslational modification of protein

Targeting and transport of the protein

Degradation of the protein

Past focus has been on understanding transcription initiation.

There is increasing elucidation of posttranscriptional and translational regulation.

Mechanisms can be elaborate and interdependent, especially in development.

Regulation relies on precise protein-DNA and protein-protein contacts.

Housekeeping gene

Regulated gene

under constitutive expression

constantly expressed in approximately all cells

Levels of the gene product rise and fall with the needs of the organism.

Such genes are inducible.
• able to be turned on

Such genes are also repressible.
• able to be turned off

RNA Polymerase Binding to Promoters Is a Major Target of Regulation

RNA polymerases bind to promoter sequences near the starting point of transcription initiation.

The RNA pol-promoter interaction greatly influences the rate of transcription initiation.

Regulatory proteins (transcription factors) work to enhance or inhibit this interaction between RNA pol and the promoter DNA.

A Consensus Sequence Is Found in Many E. Coli Promoters

Most bacterial promoters include the conserved –10 and –35 regions that interact with the σfactor of RNA polymerase.

Some promoters also include the upstream element that interacts with the αsubunit of RNA polymerase.

Mechanisms to Regulate Transcription in Bacteria

Use of σ factors

Binding other proteins (transcription factors) to promoters

recognize different classes of promoters

allows coordinated expression of different sets of genes

recognize promoters of specific genes

may bind small signaling molecules

may undergo posttranslational modifications

protein’s affinity toward DNA is altered by ligand binding or posttranslational modifications

allows expression of specific genes in response to signals in the environment

Regulation by Specificity Factors Such as σSubunits of RNA Pol
-Specificity factors alter RNA polymerase’s affinity for certain promoters.

Heat Shock Induces Transcription of New Products to Protect Cell

Occurs when bacteria are subject to heat stress

RNA Pol replaces σ70 with σ32

Causes RNA Pol to bind to different set of promoters

Small-Molecule Effectors Can
Regulate Activators and Repressors

Repressors reduce RNA Pol-promoter interactions or block the polymerase.

Effectors can bind to repressor and induce a conformational change.

Activators Improve Contacts Between
RNA Polymerase and the Promoter

Binding sites in DNA for activators are called enhancers.

In bacteria, enhancers are usually adjacent to the promoter.

In eukaryotes, enhancers may be very distant from the promoter.

Negative Regulation

Positive Regulation

DNA Looping Allows Eukaryotic Enhancers to Be Far from Promoters

Activators can influence transcription at promoters thousands of bp away.

How? Via formation of DNA loops

Looping can be facilitated by architectural regulator proteins.

Co-activators may mediate binding by binding to both activator and RNA polymerase.

Many Bacterial Genes Are Transcribed And Regulated Together in an Operon

An operon is a cluster of genes sharing a promoter and regulatory sequences.

First example: the lac operon

Lac operon as a model for regulation

The lac Operon Reveals Many
Principles of Gene Regulation

Work of Jacob and Monod − 1960

Shows how three genes for metabolism of lactose are regulated together as an operon:

β-galactosidase (lacZ):cleaves lactose to yield glucose and galactose

lactose permease (galactoside permease; lacY):transports lactose into cell

thiogalactoside transacetylase (lacA)

They rely on negative regulation via a repressor.

Lactose Metabolism in E. Coli

When glucose is abundant and lactose is lacking, cells make only very low levels of enzymes for lactose metabolism.

If glucose is scarce and cells are fed lactose, the cells can use it as their energy source.

The cells suddenly express the genes for the enzymes for lactose metabolism.

Lactose Metabolism in E. Coli

Inhibiting the Transcription of the
lac Operon via a Repressor Protein

The Lac repressor binds primarily to the operator O1.

The repressor also binds to one of two secondary operators, with the DNA looped between this secondary operator and O1

A gene called lacI encodes a repressor called the Lac repressor.

Structure of the lac Operon

Lac Repressor Bound to O1 and O3 with DNA Looped Between

The lac Operon Is Induced by Allolactose

How Lac Repressor Binds to DNA

Lac repressor is a tetramer.

The O2 sequence reflects the symmetry of the repressor.

There are approximately 20 repressors per cell.

The lac Operon Is Governed by More Than Repressor Binding

The availability of glucose governs expression of lactose-digesting genes via catabolite repression.

When glucose is present, lactose genes are turned off.

It is mediated by cAMP and cAMP receptor protein(CRP or CAP for catabolite activator protein).

When Glucose Is Absent, lac Operon Transcription Is Stimulated by CRP-cAMP

When Lactose Is Absent Little to No Transcription Occurs

When Lactose Is Present, Transcription Depends On Glucose Level

Two Requirements for Strongest Induction of the lac Operon

Lactose must be present to form allolactose to bind to the repressor and cause it to dissociate from the operator.(reducing repression )

[Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can. bind near the promoter(causing activation)

Combined Effects of Glucose and Lactose on the lac Operon

inhibition(When lactose is low, repressor is bound)

permitting transcription(When lactose is high, repressor dissociates)

transcription is dampened(When glucose is high, CRP is not bound and)

activation(When glucose is low, cAMP is high and CRP is bound)

Protein factors that control transcription

Binding of Proteins to DNA Often
Involves Hydrogen Bonding

Gln/Asn can form a specific H-bond with adenine’s N-6 and H-7 H’s.

Arg can form specific H-bonds with the cytosine-guanine base pair.

The major groove is the right size for the α helix and has exposed H-bonding groups.

Protein-DNA Binding Motifs

A few protein arrangements are used in thousands of different regulatory proteins

and are hence called motifs.

helix-turn-helix(used by Lac repressor)

zinc finger

leucine zipper

and so on

The Helix-Turn-Helix Motif Is Common in DNA-Binding Proteins

Helix-Turn-Helix Motif

The Zinc Finger Motif Is Common in
Eukaryotic Transcription Factors

~30 aa

“Finger” portion is a peptide loop cross-linked by Zn2+

Interact with DNA or RNA
-Binding is weak, so several zinc fingers often act in tandem.

Binding can range from sequence specific to random.

Zn2+usually coordinated by 4 Cys, or 2 Cys, 2 His

Zinc Fingers

The Leucine Zipper Motif

Dimer of two amphipathic αhelices plus a DNA-binding domain

Each helix is hydrophobic on one side and hydrophilic on the other.

The hydrophobic side is the contact between the two monomers.

Approximately every seventh residue in helices is Leu (L).

Helices form a coiled coil.

The DNA-binding domain has basic residues (Lys (K), Arg (R)) to interact with polyanionic DNA.

Eukaryotic Gene Regulation

Amino Acid Biosynthesis Regulated

The trp Operon

SOS Response

Synthesis of Ribosomal Proteins and
rRNA is Controlled at Translation

Translational Feedback Mechanism

rRNA Synthesis Is Also Regulated
by Amino Acid Availability

Stringent Factor Catalyzes Formation of
an Unusual Guanosine-Based Messenger

Stringent Response in E. Coli

When bacteria need more protein (as in cell growth), they make more ribosomes.

Ribosmal protein (r-protein) operons are regulated via translational feedback (next slide).

Each operon for an r-protein encodes a translational repressor.

Repressor has greater affinity for rRNA than for mRNA.

The stringent response occurs when aa concentrations are low.

Lack of aa produces uncharged tRNA.

Uncharged tRNA binds to ribosomal A site.

rRNA synthesis triggers a cascade that begins with binding stringent factor protein (RelA) to ribosome.

Stringent factor catalyzes formation of nucleotide guanosine tetraphosphate (ppGpp).

Binding of ppGpp to RNA polymerase reduces rRNA synthesis.

其他

Activation of Bacterial Translation
by Small RNA Molecules

Inhibition of Bacterial Translation
by Small RNA Molecules

Regulation of Bacterial mRNA Function in Trans by sRNA

Cis Regulation by Riboswitches

Trans-Acting sRNAs Facilitate
Translation of σS mRNA

Riboswitches Are a Developing
Area of Research

Some RNAs Participate in Regulation

“Cis” regulation: a molecule affects its own function

“Trans” regualtion: a molecule is affected by another separate molecule

σS mRNA is present a low levels but is not translated due to a hairpin structure that inhibits its binding to ribosomes.

sRNAs (small RNAs) bind to this mRNA and inhibit formation of the hairpin.

sRNA-mRNA interactions are facilitated by a chaperone protein called Hfq.

The ribosome-binding Shine−Dalgarno sequence is sequestered into a stem-loop structure in the mRNA.

In the presence of protein Hfq, small regulatory RNA DsrA binds to the mRNA.

The binding of DsrA opens up the stem-loop and allows mRNA binding to the ribosome.

DsrA RNA promotes translation.

The ribosome-binding Shine−Dalgarno sequence is sequestered into a stem-loop structure in the mRNA.

In the presence of protein Hfq, small regulatory RNA OxyS binds to the mRNA.

The binding of OxyS blocks the ribosome binding site in mRNA.

OxyS RNA inhibits translation.

Riboswitches have been found to respond to many coenzymes, metabolites, and so on.

They are also found in eukaryotic introns and seem to regulate splicing.

Some riboswitches are unique to bacteria and are therefore a target for antibiotics.

Three Features of Transcriptionally
Active Chromatin

Nucleosomes Can Be Restructured by Specific Protein Complexes

Features of Eukaryotic Gene Regulation

Histone Modification Alters Transcription

Phase Variation – Regulation of Flagellin Genes

Covalent Modification of Histones

Regulation by Gene Recombination

Nucleosome Remolding

Processes can remove promoters relative to the coding sequence or can put genes into multiple orientations to alter the expression.

Example: Flagellin genes in Salmonella

In one orientation fljB is expressed with a repressor for fljC gene.

In another orientation, only fljC is expressed.

The process is called phase variation.

Access of eukaryotic promoters to RNA polymerase is hindered by chromatin structure.

Positive regulation mechanisms predominate and are required for even a basal level of gene expression.

Eukaryotic gene expression requires a complicated set of proteins.

Euchromatin = less-condensed chromatin, distinguished from transcriptionally inactive heterochromatin

Chromatin remodeling of
transcriptionally active genes:

nucleosomes repositioned

histone variants

covalent modifications to nucleosomes

Methylation

Phosphorylation

Acetylation

Ubiquitination

Sumoylation

Occur mostly in the N-terminal domain of the histones found near the exterior of the nucleosome particle

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