Chapters 18 - Regulation of gene expression in eukaryotes:

Levels of control of gene expression

Short term control
(to meet the daily needs of the organism)
Long term control
(gene regulation in development/differentiation)

Differences between prokaryotes and eukaryotes:

Prokaryote gene expression typically is regulated by an operon, the

collection of controlling sites adjacent to polycistronic proteincoding sequences.

Eukaryotic genes also are regulated in units of protein-coding

sequences and adjacent controlling sites, but operons are not known
to occur.

Eukaryotic gene regulation is more complex because eukaryotes

possess a nucleus.
(transcription and translation are not coupled).

Two categories of eukaryotic gene regulation exist:

Short-term - genes are quickly turned on or off in response to the
environment and demands of the cell.
Long-term - genes for development and differentiation.

Eukaryote gene expression is regulated at six levels:

1.

Transcription

2.

RNA processing

3.

mRNA transport

4.

mRNA translation

5.

mRNA degradation

6.

Protein degradation

Fig. 18.1

1. Transcription control of gene regulation is controlled by:

1.

Promoters

Occur upstream of the transcription start site.

Some determine where transcription begins (e.g., TATA),

whereas others determine if transcription begins.

Promoters are activated by specialized transcription factor (TF)

proteins (specific TFs bind specific promoters).

One or many promoters (each with specific TF proteins) may

occur for any given gene.

Promoters may be positively or negatively regulated.

1. Transcription control of gene regulation is controlled by:

2.

Enhancers

Occur upstream or downstream of the transcription start site.

Regulatory proteins bind specific enhancer sequences; binding

is determined by the DNA sequence.

Loops may form in DNA bound to TFs and make contact with
upstream enhancer elements.

Interactions of regulatory proteins determine if transcription is

activated or repressed (positively or negatively regulated).

Fig. 18.2
Activation of transcription
By transcription factors (TFs),
activator, and coactivator
proteins.

More about promoters and enhancers:

Some regulatory proteins are common in all cell types, others are
specific.

Each promoter and enhancer possesses a specific set of proteins

(coactivators) that determines expression.

Rate of gene expression is controlled by interaction between positive

and negative regulatory proteins.

Combinatorial gene regulation; enhancers and promoters bind many

of the same regulatory proteins, implying lots of interaction with fine
and coarse levels of control.

Chromosome structure, eukaryote chromosomes are packed with histones:

Prokaryotes lack histones and other structural proteins, so access to the DNA
is straightforward.

Eukaryotes possess histones, and histones repress transcription because they

interfere with proteins that bind to DNA.

Verified by DNase I sensitivity experiments:

DNase I readily degrades transcriptionally active DNA.

Histones shield non-transcribed DNA from DNase I, and DNA does not
degrade as readily.

If you experimentally add histones and promoter binding proteins; histones

competitively bind to promoters and inhibit transcription.

Transcriptionally active genes possess looser chromosome structures than

inactive genes.

Histones are acetylated and phosphorylated, altering their ability to bind

to DNA.

Enhancer binding proteins competitively block histones if they are added

experimentally with histones and promoter-binding TFs.

RNA polymerase and TFs step-around the histones/nucleosomes and

transcription occurs.

Fig. 18.10a
Chromatin remodeling
Acetylation of histones enhances
access to promoter region and
facilitates transcription.

DNA methylation and transcription control:

Small percentages of newly synthesized DNAs (~3% in mammals)

are chemically modified by methylation.

Methylation occurs most often in symmetrical CG sequences.

Transcriptionally active genes possess significantly lower levels of

methylated DNA than inactive genes.

A gene for methylation is essential for development in mice

(turning off a gene also can be important).

Methylation results in a human disease called fragile X

syndrome; FMR-1 gene is silenced by methylation.

Fig. 18.12

Fig. 18.13
Methylation of H19 inactivates transcription
(involved in expression of insulin like growth factor)

Chromatic remodeling and DNA methylation are the basis

for epigenetic inheritance.

Short-term - transcriptional control of galactose-utilizing genes in yeast:

3 genes (GAL1, GAL7, & GAL 10) code enzymes that function in the
galactose metabolic pathway.

GAL1

galactokinase

GAL7

galactose transferase

GAL10

galactose epimerase

Pathway produces d-glucose 6-phosphate, which enters the

glycolytic pathway and is metabolized by genes that are
continuously transcribed.

In absence of galactose, GAL genes are not transcribed.

GAL genes rapidly induced by galactose and absence of glucose.

Analagous to E. coli lac operon repression by glucose.

Galactose metabolizing pathway of yeast.

Short-term - transcriptional control of galactose-utilizing genes in yeast:

GAL genes are near each other but do not constitute an operon.

Additional unlinked gene, GAL4, codes a repressor protein that binds

a promoter element called an upstream activator sequence (UASG).

UASG is located between GAL1 and GAL10.

Transcription occurs in both directions from UASG.

When galactose is absent, the GAL4 product (GAL4p) and another

product (GAL80p) bind the UASG sequence; transcription does not
occur.

When galactose is added, a galactose metabolite binds GAL80p and

GAL4p amino acids are phosphorylated.

Galactose acts as an inducer by causing a conformation change in

GAL4p/GAL80p.

Activation model of GAL genes in yeast.

Fig. 18.4b

Fig. 18.4c

Hormone regulation - another good example of short-term regulation of

transcription:

Cells of higher eukaryotes are specialized and generally shielded

from rapid changes in the external environment.

Hormone signals are one mechanism for regulating transcription in

response to demands of the environment.

Hormones act as inducers produced by one cell and cause a

physiological response in another cell.

Hormones act only on target cells with hormone specific receptors,

and levels of hormones are maintained by feedback pathways.

Hormones deliver signals in two different ways:

Steroid hormones pass through the cell membrane and bind

cytoplasmic receptors, which together bind directly to DNA
and regulate gene expression.

Polypeptide hormones bind at the cell surface and activate

transmembrane enzymes to produce second messengers
(such as cAMP) that activate gene transcription.

Examples of mammalian steroid hormones.

Steroid hormones are four ring structures/differences derive
from differences in side-groups.

Plant hormones.

Hormone regulation continued:

Genes regulated by steroid hormones possess binding regions in

the sequence called steroid hormone response elements (HREs).

HREs often occur in multiple copies in enhancer sequence regions.

When steroid is absent:

receptor is bound and guarded by chaperone proteins;
transcription does not occur.

When steroid is present:

Steroid displaces the chaperone protein, binds the receptor,
and binds the HRE sequence; transcription begins.

Fig. 18.17, Model of glucocorticoid steroid hormone regulation.

2. RNA processing control:

RNA processing regulates mRNA production from precursor RNAs.

Two independent regulatory mechanisms occur:

Alternative polyadenylation

= where the polyA tail is added

Alternative splicing

= which exons are spliced

Alternative polyadenylation and splicing can occur together.

Examples:

Human calcitonin (CALC) gene in thyroid and neuronal cells

Sex determination in Drosophila

Fig. 18.14, Alternative polyadenylation and splicing of the human

CACL gene in thyroid and neuronal cells.

Calcitonin
gene-related
peptide

Fig. 19.13
Alternative splicing in
sex determination of
Drosophila
Sex is determined by
X:A ratio.
Sxl (sex lethal) gene
determines the
pathways for males
and females.
If X:A = 1, all introns
and exon 3 (which
contains the stop
codon) are removed.
If X:A = 0.5, no
functional protein is
produced.

3. mRNA transport control:

Eukaryote mRNA transport is regulated.

Some experiments show ~1/2 of primary transcripts never leave the

nucleus and are degraded.

Mature mRNAs exit through the nuclear pores.

4. mRNA translation control:

Unfertilized eggs are an example, in which mRNAs (stored in the

egg/no new mRNA synthesis) show increased translation after
fertilization).

Stored mRNAs are protected by proteins that inhibit translation.

Poly(A) tails promote translation.

Stored mRNAs usually have short poly(A) tails

(15-90 As vs 100-300 As).

Specific mRNAs are marked for deadenylation (tail-chopping) prior

to storage by AU-rich sequences in 3-UTR.

Activation occurs when an enzyme recognizes AU-rich element and

adds ~150 As to create a full length poly(A) tail.

5. mRNA degradation control:

All RNAs in the cytoplasm are subject to degradation.

tRNAs and rRNAs usually are very stable; mRNAs vary considerably
(minutes to months).

Stability may change in response to regulatory signals and is

thought to be a major regulatory control point.

Various sequences and processes affect mRNA half-life:

AU-rich elements

Secondary structure

Deadenylation enzymes remove As from poly(A) tail

5 de-capping

Internal cleavage of mRNA and fragment degradation

6. Post-translational control - protein degradation:

Proteins can be short-lived (e.g., steroid receptors) or long-lived

(e.g., lens proteins in your eyes).

Protein degradation in eukaryotes requires a protein co-factor called

ubiquitin. Ubiquitin binds to proteins and identifies them for
degradation by proteolytic enzymes.

Amino acid at the N-terminus is correlated with protein stability and

determines rate of ubiquitin binding.

Arg, Lys, Phe, Leu, Trp

1/2 life 3 minutes

Cys, Ala, Ser, Thr, Gly, Val, Pro, Met

1/2 life 20 hours

Summary and contrasts:

Prokaryotes control expression by:
Transcription
Eukaryotes control expression by:
Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation

Fig. 18.1