LECTURE 19: RNA REGULATORY MECHANISMS

1. 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.

2. 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.

3. BLZ Factors are Another Class of Homomeric DNA Binding Proteins

4. Homeodomain-Containing Factors Contain Helix-Turn-Helix Motif Heterodimerization provides greater target specificity by lengthening recognition sequence.

5. Multiple Zinc Fingers Allow Complex Sequence Recognition

6. Prokaryotic Regulation by Induction and Repression of Transcription Initiation Lactose catabolism utilizes regulation at the LAC operon

7. 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

8. 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

9. 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)

10. 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.

11. 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

12. 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.

13. 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.

14. 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.

15. 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.

16. Hormone Nuclear Receptors Have Ligand-Binding and DNA Binding Domains

17. Ligand Binding Enables Receptors To Recruit Coactivators

18. 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.

19. 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.