Molecular Mechanisms of Gene Regulation

1. Molecular Mechanisms of Gene Regulation Genetics Spring 2014

2. Outline

3. Transcriptional Regulation in Prokaryotes Genes are never really completely off, basal level of transcription exits. Random bursts of gene expression, stochastic noise exists as well. Bacterial mRNA is polycistronic (Operon) allowing for coordinate regulation of genes. Polycistronic mRNA; ribosomes can initiate at internal sites in mRNA. Monocistronic mRNA; ribosomes cannot initiate at internal sites in mRNA.

4. Positive regulation: default state is ‘off’; activator turns on Negative regulation: default state is ‘on’; repressor turns off Lac operon and Crp protein: Cyclic AMP controls catabolism, cell senses level of glucose Inducible systems (lac operon): often catabolic, degrade carbon sources Repressible systems (trp operon): often anabolic, synthesize amino acids

5. Transcriptional Regulation in Prokaryotes – The lac Operon In E.coli, the system responsible for the degradation of the sugar lactose is the lac operon, with negative regulation and inducible transcription. • lacI encodes repressor • lacO is the lac operator where repressor binds • lacP is the promoter • lacZ, Y, and A are structural genes

7. The lac operon is regulated by the lac repressor (negative regulation) and cAMP-CRP (positive regulation) -lactose + glucose - lactose - glucose + lactose - glucose + lactose + glucose

8. Sequences in the lac promoter region How do lactose and glucose function together to regulate the transcription of the lac operon? Structure of the lac operon repression loop where the repressor protein (violet) binds to with two operator regions (Op3 and Op1 in red) to form a loop that the DNA sequence at which CRP binds. In this configuration, is the lac operon transcribed?

9. Transcriptional Regulation in Prokaryotes – The trp Operon Structure of the trp operon is similar to that of the lac operon. This system is repressible, anabolic for synthesis of tryptophan‘on’ unless not needed, then ‘off’.

10. Trp repressor requires tryptophan as corepressor‘on’ unless not needed, then ‘off’

11. Regulation through Transcription Termination Attenuation: Some tryptophan is present but not enough to sustain optimal growth, therefore cells need to synthesize tryptophan BUTnot at maximum level (fine tuning). Fine tunes anabolic operons, senses levels of charged tRNA-trp and level of translation; depends on alternate conformation of trp leader mRNA

12. Riboswitch regulation of transcription termination by the yitJ leader RNA in Bacillus subtilis Embedded in the 5’ untranslated region preceding the gene-coding sequence of an mRNA transcript, the riboswitch naturally folds into a well-defined three-dimensional structure.

13. Transcriptional Regulation in Eukaryotes While housekeeping genes are expressed at a constitutively low rate, other genes need to be regulated expression involves cumulative actions: • Transcriptional activator proteins (positive regulation, trans-acting). • Binding enhancer DNA sequences (cis-acting). • Differential splicing. • Protein factors cause silencing of transcription (negative regulation, trans-acting). • Chromatinstructure affects expression (chromatin remodeling proteins).

14. How cis-acting and trans-acting elements influence transcription

15. Transcriptional Regulation in Eukaryotes - yeast The earliest model systems for studying transcriptional regulation was that of the galactose-mediated induction of gene expression in yeast. Metabolic pathway by which galactose is converted to glucose-1-phosphate in the yeast Saccharomyces cerevisiae. What are the differences and similarities between transcriptional regulation in bacteria and yeasts?

16. More specifically the yeast Gal4 protein has been used as a model for studying transcriptional regulation in eukaryotes. The nuclear protein GAL4 is a positive regulator of gene expression for the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1. GAL4 recognizes a 17 base-pair long sequence in the upstream activating sequence (uas) of these genes, GAL4 binds to the DNA as a homodimer.

17. Transcriptional Regulation in Eukaryotes – Enhancers and Silencers • Enhancer and silencer sequences • Short (usually <20 bp) • Located upstream, downstream, or within gene • Function in regular or in inverted orientation • Bind specific proteins (regulatory transcription factors; activator and repressor) Hormonal responsiveness of integrated MMTV DNA: Positions, in the Mouse Mammary Tumor Virus, of enhancers that allow transcription of the viral sequence to be induced by glucocorticoid steroid hormone. LTR stands for Long Terminal Repeat.

18. Transcriptional Regulation in Eukaryotes - Transcriptome • Transcriptional activatorsrecruit the transcription machinery. • (pol II and basal or general transcription factors) • TFIID = TBP + TAFs • Many enhancers activate transcription by DNA looping

19. Transcriptome: Multiple enhancers and activators can regulate a eukaryotic promoter Transcriptional activation during Drosophila development; TFIID has many subunits (TAFs) which are contacted by different transcription factors.

20. Transcriptional Regulation in Eukaryotes – Chromatin-Remodeling Complex (CRCs) • Nucleosomes may conceal protein-binding sites on DNA. • Chromatin remodeling complexes use ATP to help expose these sites.

21. Alternative promoters permit synthesis of different RNA or protein products such as in the transcription of alcohol dehydrogenase in Drosophila.

22. Regulation by competition for an enhancer where promoter activation depends on which activator binds to the enhancer.

23. Epigenetic Mechanisms of Transcriptional Regulation • Hereditable changes not due to changes in DNA sequence, but ‘ in addition to’ the DNA sequence. • The chemical modifications include cytosine methylation especially in 5’-CpG-3’ sequences.

24. The methylated cytosines in CCGG sequences can be detected by cleavage with restriction enzymes. Here, MspI cleaves methylated DNA while HpaII cannot

25. Characteristics of Genomic Imprinting • Imprinting occurs in the germ line • It is accompanied by heavy methylation • Imprinted genes are differentially methylated in the female and male germ lines • Once imprinted and methylated, a silenced gene remains transcriptionally inactive during embryogenesis • Imprints are erased early in germ-line development, then later re-established according to sex-specific patterns

27. RNA Interference When altered, miRNA can contribute to cancer such as colon cancer Self-assembled microsponges of hairpin RNA polymers achieve, with one thousand times lower concentration, the same degree of gene silencing in tumour-carrying mice as conventional nanoparticle-based siRNA delivery vehicles.

28. Programmed DNA Rearrangements Programmed DNA rearrangements expand repertoire of proteins: • Gene amplification in toad Xenopuslaevis yields millions of copies of rRNA and tRNA genes. • Antibody and T-cell receptor diversity arises from genomic DNA specifically cut and pasted in precursors of B and T cells with random choices of motifs from V, J regions. • Mating type switch in yeast involves movement of DNA cassettes to active mating locus plus interaction of mating factors; special HO enzyme. • light chain • 250 V regions • 4 J regions • 250 x 4 = 1000 • heavy chain • 250 V regions • 10 D regions • 4 J regions • 250 x 10 x 4 = 10,000 • 1000 light chains x 10000 heavy chains = 107 possibilities

30. Review Problems