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LECTURE 19: RNA REGULATORY MECHANISMS
Levels of specific messenger RNAs can differ in different types of cells
and at different times in the same cell.
In prokaryotes, control of RNA abundance can be at the level of
transcription initiation or transcription elongation (attenuation).
In eukaryotes, RNA abundance is regulated by transcription initiation,
attenuation, splicing, or rate of RNA degradation.
Control of transcription initiation in eukaryotes is exerted in gene-specific
manner by sequence-specific DNA binding proteins and in a region- or
chromosome-wide manner by covalent modifications of DNA or chromatin.
Sequence Specific Homodimeric DNA Binding Proteins Bind DNA Palindromes
Protein sides chains often achieve binding specificity by hydrogen bonding
with base motifs within the DNA major groove.
BLZ Factors are Another Class of Homomeric DNA Binding Proteins
Homeodomain-Containing Factors Contain Helix-Turn-Helix Motif
Heterodimerization provides greater target specificity by lengthening
recognition sequence.
Multiple Zinc Fingers Allow Complex Sequence Recognition
Prokaryotic Regulation by Induction and Repression of Transcription Initiation
Lactose catabolism utilizes regulation at the LAC operon
Lac Operon Encodes Multiple Proteins Involved in Lactose Metabolism
Lac repressor is constitutively synthesized from the LacI gene
Repressor binds to operator (LacO) just downstream of operator promotor
and prevents operon transcription by blocking RNA polymerase movement
Lactose or non-hydrolizable analogs bind the repressor and prevent
its binding to LacO, thereby promoting operon transcription
Glucose Inhibits Lac Operon Transcription Initiation by Catabolite Repression
Efficient binding of RNA polymerase to the
Lac operon promoter (P) requires binding of
cAMP to CAP proteins.
cAMP accumulates only when ATP levels are
low. Glucose, the preferred energy source,
enables ATP synthesis and drop in cAMP
level, thereby inhibiting transcription
of the Lac operon
Prokaryotic Attenuation Enables Amino Acids to Inhibit Expression
of Amino Acid Biosynthetic Enzymes
TRP attenuator is preceding by short ORF with TRP codons
In absence of charged TRP tRNA, ribosome stalls at TRP codons and
prevents attenuator RNA region from adopting a stem-loop mediating
transcription termination (attenuation)
Eukaryotic DNA Is Compacted by Assembly Into Nucleosomes
Histone proteins H2A, H2B, H3, H4 have basic (+ charged) tails and
complex into a core octamer. DNA wraps around the core, stabilized by
electrostatic interaction between histone tails and
backbone phosphates along DNA chain.
Each nucleosome contains 140 bp DNA, with an
internucleosomal distance usually 40 bp.
Tight binding of DNA to histones in nucleosomes
inhibits access of transcription factors, repressing
transcription.
Histone Acetylation Helps Activate Chromatin
Acetylation of histone tails in nucleosomes weakens DNA binding to
histone cores, creating “open” chromatin competent for gene transcription.
Open chromatin is amenable to transcription factor binding to specific
sites on DNA (promoters/ enhancers)
Acetylated histones also directly recruit other proteins
containing bromodomains, some of which have
activator regions that help assemble transcription
machinery.
Non-acetylated chromatin leads to other modifications
(e.g., CpG DNA methylation) that stabilizes inactive state
X Chromosome Inactivation Is Mammalian Mechanism for Control of Gene Dosage
In most tissues, gene expression must be precisely controlled. X chromosome
presents a particular developmental challenge, since males have one X and
females have two X chromosomes. Problem solved by X inactivation.
Random X Inactivation Mediated by Xic, Xist, and Tsix
Random X chromosome inactivation mediated by interaction of X inactivation
centers (Xics) on two X chromosomes of female cells.
Xic interaction first triggers transcription of Xist and Tsix RNAs from each
X chromosome. Xist and Tsix are large RNAs without coding sequences.
Xist binds to the chromosome from which it was transcribed, and Tsix
probably prevents accidental binding to other X chromosome.
Xist RNA eventually “paints” the entire X chromosome from which it is
transcribed, causing inactivation …. By this point, the only gene
transcribed from the inactivated chromosome is Xist.
Meanwhile, Tsix paints the active X chromosome, preventing spread of
inactivation to the second chromosome.
Mechanism of Xist-Mediated X Inactivation Still Uncertain
Xist RNA recruits novel proteins to inactivating X chromosome, induces
methylation of histone tails, and bears a 5’ end essential for gene
silencing.
After induction of X-inactivation, Xist is no longer required for its maintenance.
Some Eukaryotic Transcription Factors Are Master Regulators of Differentiation
Myogenin and MyoD are bHLH transcription factors that drive differentiation
of skeletal muscle. All genes encoding proteins specific for muscle have
enhancers that recruit Myogenin and MyoD.
Ectopic expression of Myogenin or MyoD in fibroblasts converts them to
muscle cells.
Homeotic proteins contain DNA-binding homeodomains. The repertoire
of homeotic proteins expressed in a region of developing embryo
determines which kind of body part will form from that tissue.
Homeotic proteins do not specify particular differentiated cell types, but
determine the shapes of developing tissue regions.
Hormone Nuclear Receptors Have Ligand-Binding and DNA Binding Domains
Ligand Binding Enables Receptors To Recruit Coactivators
Nuclear Hormone Receptors Classified Into Two Subgroups
Based Upon Behavior of Receptor in Absence of Hormone
(Subgroup 1)
One subclass of nuclear receptors includes glucocorticoid, estrogen,
and progesterone receptors.
In absence of hormone, the hormone-binding domain interacts with
Hsp90 (a heat shock protein)
Binding of Hsp90 prevents the receptor from docking to its DNA
recognition site. Mechanism is probably a steric obstruction by the
large Hsp90 protein
Hormone must have greater affinity for receptor than does Hsp90,
thereby displacing Hsp90 and allowing for DNA binding along
with co-activator recruitment.
Nuclear Hormone Receptors Classified Into Two Subgroups
Based Upon Behavior of Receptor in Absence of Hormone
(Subgroup 2)
Second subclass of receptors includes those for thyroid hormone,
retinoic acid, and vitamin D.
In absence of hormone, these receptors do not bind Hsp90 and still
associate with their sequence-specific DNA binding sites.
Without hormone, docked receptors cannot recruit co-activators.
Unliganded receptors, in fact, recruit repressor proteins or protein
complexes, including histone deacetylases (HDACs), which render
regional chromatin inactive through removal of histone acetylations.
Gene expression regulated by these receptors is under very tight control,
mediating both repression and activation of transcription.