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