Transcript CHAPTER 12

Transcriptional-level control (10)
• Researchers use the following techniques to
find DNA sequences involved in regulation:
– Deletion mapping
– DNA footprinting
– Genome-wide location analysis
• Allows simultaneous monitoring of all the sites within
the genome that carry a particular activity.
Use of chromatin
immunoprecipitation
to identify transcription
factor-binding sites
Transcriptional-level control (11)
• The Glucocorticoid Receptor: An Example of
Transcriptional Activation
– PEPCK is a key enzyme controlled by a variety of
transcription factors called response elements.
Transcriptional-level control (12)
• The Glucocorticoid Receptor (continued)
– The glucocorticoid receptor (GR) is a nuclear
receptor that includes a ligand-binding domain
and a DNA-binding transcription factor.
– The GR binds to a glucocorticoid response element
(GRE), which is a palindrome.
Activation of a gene by a steroid hormone
Transcriptional-level control (13)
• Transcriptional Activation: The Role of
Enhancers, Promoters, and Coactivators
– Enhancers are DNA elements that stimulate
transcription.
• Can be located very far upstream from the regulated
gene.
• A promoter and its enhancers can be “cordoned off”
from other elements by sequences called insulators.
A survey of transcriptional activation
Transcriptional-level control (14)
• Coactivators serve as intermediates for
transcription factors, and are divided into two
classes:
– Those that interact with the transcription
machinery.
– Those that alter chromatin structure modifying
histones to regulate transcription.
• By using histone acetyltransferases (HATS)
• By using chromatin remodeling complexes
Selective localization of histone modifications
A model of events following
the binding of a
transcriptional activator
Several alternative actions of chromatin
remodeling
The nucleosomal landscape of yeast genes
Transcriptional-level control (15)
• Transcriptional Activation from Poised
Polymerases
– RNA polymerases are also bound to
“transcriptionally silent” genes that initiate
transcription but do not transition to elongation.
– These polymerases are ready for transcription but
are poised by inhibitory factors.
– Gene transcription at the level of elongation may
be important in activation of genes.
Transcriptional-level control (16)
• Transcriptional Repression
– Histone deacetylases (HDACs) remove acetyl
groups and repress transcription.
• HDACs are subunits of larger complexes acting as
corepressor.
• Corepressors are recruited to specific gene loci by
transcription factors that cause the targeted gene to be
silenced.
A model for
transcriptional
repression
Transcriptional-level control (17)
• Transcriptional Repression (continued)
– DNA Methylation
• It is carried out by DNA methylatransferases.
• It silences transcription in eukaryotic cells.
• Methylation patterns of gene regulatory regions change
during cellular differentiation.
Transcriptional-level control (18)
• Transcriptional Repression (continued)
– DNA Methylation and Transcriptional Repression
• Activity of certain genes varies according to changes in
DNA methylation.
• DNA methylation serves more to maintain a gene in an
inactive state rather than to initially inactivate it.
• DNA methylation is not an universal mechanism for
inactivating eukaryotic genes.
Changes in DNA methylation levels during
mammalian development
Transcriptional-level control (19)
• Transcriptional Repression (continued)
– Genomic Imprinting
• Activity of certain genes, called imprinted genes,
depends on whether they originated with the sperm or
egg.
• Active and inactive versions of imprinted genes differ in
their methylation patterns.
• Disturbances in imprinting patterns have been
implicated in a number of rare human genetic
disorders.
12.5 Processing-level Control (1)
• Protein diversity can
be generated by
alternative splicing.
• Alternative splicing
can become
complex, allowing
different
combinations of
exons in the final
mRNA product.
Processing-level Control (2)
• There are factors
that can influence
splice site selection.
• Exonic splicing
enhancers serve as
binding sites for
regulatory proteins.
Processing-level Control (3)
• RNA Editing
– Specific nucleotides can be converted to other
nucleotides through mRNA editing.
– RNA editing ca create new splice sites, generate
stop codons, or lead to amino acid substitutions.
– It is important in the nervous system, where
messages need to have A converted to I (inosine)
to generate a glutamate receptor.
12.6 Translational-level Control (1)
• Translation of mRNAs that have been
transported from the nucleus to the
cytoplasm is regulated.
– Translational-level control occurs via interactions
of specific mRNAs and proteins in the cytoplasm.
– Regulatory proteins act on unstranslated regions
(UTRs) at both their 5’ and 3’ ends.
– UTRs contain nucleotide sequences used by the
cell to mediate translational-level control.
Translational-level Control (2)
• Cytoplasmic Localization of mRNAs
– In the fruit fly embryo the development of
anterior-posterior axis is regulated by the
localization of specific mRNAs along the axis in the
egg.
– Cytoplasmic localization of mRNAs is determined
by their 3’ UTRs.
Cytoplasmic localization of mRNAs
Cytoplasmic localization of mRNAs
Translational-level Control (3)
• The Control of mRNA Translation
– Several important processes depend on mRNAs
that were synthesized at a previous time and
stored in the cytoplasm in an inactive state.
– Other mechanisms influence the rate of
translation of specific mRNAs through proteins
that recognize specific elements in the UTRs of
those mRNAs.
– Example: mRNA that codes for ferritin.
A model for the mechanism of translational
activation of mRNAs following fertilization
Translational-level Control (4)
• The Control of mRNA Stability
– The lifetimes of eukaryotic mRNA vary widely.
– Poly(A) tail length may influence the longevity of
mRNA.
• As an mRNA remains in the cytoplasm, its poly(A) tail
tends to be reduced.
• When the tail is about 30 A residues, the tail is
shortened.
– Certain destabilizing proteins in the 3’ UTR may
affect the rate of poly(A) tail shortening.
mRNA degradation in mammalian cells
Translational-level Control (5)
• The Control of mRNA Stability (continued)
– Deadenylation, decapping, and 5’ 3’
degradation occur within small transient
cytoplasmic granules (P-bodies).
– P-bodies can also store mRNAs no longer being
translated.
Translational-level Control (6)
• The Role of MicroRNAs in Translational-level
Control
– miRNAs act by binding to site in the 3’UTR of their
target mRNAs.
– Translational-level by miRNAS has been a bit
controversial.
– Some studies suggest that miRNAs carry out
translational-level control by inducing the
degradation of target mRNA.
Potential mechanisms by which miRNAs might
decrease gene expression at translational level
12.7 Post-translational Control:
Determining Protein Stability (1)
• The factors that control a protein’s lifetime are
not well understood.
• Protein stability may be determined by the
amino acids on the N-terminus.
• Degradation of proteins is carried out within
hollow, cylindrical proteasomes.
Post-translational Control:
Determining Protein Stability (2)
• Proteasomes recognize proteins linked to
ubiquitin.
• Ubquitin is transferred by ubiquitin ligases to
proteins being degraded.
• Once polubiquitanated, a protein is
recognized by the cap of the proteasome.
• Once degraded, the component amino acids
are released back into the cytosol.
Proteasome structure
Proteasome structure