Chapter 11: Transcription in Eukaryotes

1. Chapter 11: Transcription in Eukaryotes

2. … the modern researcher in transcriptional control has much to think about. James T. Kadanoga, Cell (2004), 116:247

3. 11.1 Introduction

4. Eukaryotic gene regulation involves: • DNA-protein interactions • Protein-protein interactions • Chromatin structure • Nuclear architecture • Cellular compartmentalization

5. 11.2 Overview of transcriptional regulation

6. Transcription and translation are uncoupled in eukaryotes • Transcription takes place in the nucleus and translation takes place in the cytoplasm. • The whole process may take hours, or in some cases, months for developmentally regulated genes. • Gene expression can be controlled at many different levels.

7. Transcription is mediated by: • Sequence-specific DNA-binding transcription factors. • The general RNA polymerase II (RNA pol II) transcriptional machinery. • Coactivators and corepressors. • Elongation factors.

8. Chromosomal territories and transcription factories • Chromosome “painting” has shown that each chromosome occupies its own distinct territory in the nucleus. • Transcription decondenses chromatin territories.

9. The DNA loops that form in decondensed regions are proposed to be associated with transcription “factories.” • Transcriptionally active genes also appear to be preferentially associated with nuclear pore complex.

10. Eukaryotes have different types of RNA polymerase • Bacteria have one type of RNA polymerase that is responsible for transcription of all genes. • Eukaryotes have multiple nuclear DNA-dependent RNA polymerases and organelle-specific polymerases. • Focus here on regulation of transcription of protein-coding genes by RNA polymerase II.

11. 11.3 Protein-coding gene regulatory elements

12. The big picture: • Transcription factors interpret the information present in gene promoters and other regulatory elements and transmit the appropriate response to the RNA pol II transcriptional machinery. • What turns on a particular gene in a particular cell is the unique combination of regulatory elements and the transcription factors that bind them.

13. Regulatory regions of unicellular eukaryotes such as yeast are usually only composed of short sequences located adjacent to the core promoter. • Regulatory regions of multicellular eukaryotes are scattered over an average distance of 10 kb of genomic DNA.

14. Variation in: • Whether a particular element is present or absent. • The number of distinct elements. • Their orientation relative to the transcriptional start site. • The distance between them.

15. Gene regulatory elements are specific cis-acting DNA sequences that are recognized by trans-acting transcription factors. • Two broad categories of cis-acting regulatory elements. • Promoter elements. • Long-range regulatory elements.

16. Structure and function of promoter elements The gene promoter is the collection of cis-regulatory elements that are: • Required for the initiation of transcription. • Increase the frequency of initiation only when positioned near the transcriptional start site. • The recognition site for RNA pol II general transcription factors.

17. The gene promoter region • Core promoter elements. • Proximal promoter elements.

18. Core promoter elements • Approximately 60 bp DNA sequence overlapping the transcription start site. • Serves as the recognition site for RNA pol II and the general transcription factors. • All core promoter elements, except for BRE, are recognized by TFIID. • A particular core promoter many contain some, all, or none of the common motifs.

19. The TATA box • First core promoter element identified in a eukaryotic protein-coding gene. • Key experiment by Pierre Chambon and colleagues demonstrated that a viral TATA box is both necessary and sufficient for specific initiation of transcription by RNA pol II in vitro. • Sequence database analysis suggests the TATA box is present in only 32% of potential core promoters.

20. Promoter proximal elements • Regulation of TFIID binding to the core promoter in yeast depends on an upstream activating sequence (UAS). • Multicellular eukaryotic genes are likely to contain several promoter proximal elements. e.g. CAAT box and the GC box

21. Promoter proximal elements • Transcription factors that bind promoter proximal elements do not always directly activate or repress transcription. • Transcription factors may serve as “tethering elements.”

22. Structure and function of long-range regulatory elements • Additional regulatory elements in multicellular eukaryotes that can work over distances of 100 kb or more from the gene promoter.

23. Enhancers and silencers • Insulators • Locus control regions (LCRs) • Matrix attachment regions (MARs)

24. Enhancers and silencers • Usually 700 to 1000 bp or more away from the start of transcription. • Increase or repress gene promoter activity either in all tissues or in a regulated manner. • Typically contain ~10 binding sites for several different transcription factors. • How can you tell an enhancer from a promoter?

25. Insulators • Chromatin boundary markers. • Enhancer or silencer blocking activity. • Insulator elements are recognized by specific DNA-binding proteins.

26. Locus control regions (LCRs) • Organize and maintain a functional domain of active chromatin. • Prototype LCR characterized in the mid-1980s as a cluster of DNase I-hypersensitive sites upstream of the -globin gene cluster.

27. -globin gene LCR is required for high-level transcription • Physiological levels of expression of the embryonic, fetal, and adult -globin genes only occurs when they are downstream of the LCR. • The DNase I hypersensitive sites contain clusters of transcription factor-binding sites and interact via extensive protein-DNA and protein-protein interactions.

28. Hispanic thalassemia and DNase I hypersensitive sites • Analysis of patients with -thalassemia has led to significant advances in understanding of the LCR of the -globin gene locus. • Partial or complete deletion of the LCR leads to reduced amounts of hemoglobin in the blood.

29. Hispanic thalassemia • ~35 kb deletion of the LCR. • The Hispanic locus is transcriptionally silent. • The entire region of the -globin gene cluster is DNase I-resistant.

30. Analysis of DNase I sensitivity • Transcriptionally active genes are more susceptible to deoxyribonuclease (DNase I) digestion.

31. Matrix attachment regions (MARs) • Organize the genome into loop domains. • Typically AT rich sequences located near enhancers in 5′ and 3′ flanking sequences. • Confer tissue specificity and developmental control of gene expression. • “Landing platform” for transcription factors. • Attach to the nuclear matrix.

32. Position effect and long-range regulatory elements • The function of many long-range regulatory elements was confirmed by their effect on gene expression in transgenic animals. • Protect transgenes from the negative or positive influences exerted by chromatin at the site of integration.

33. Position effect • Expression of a transgene is unpredictable in a transgenic organism. • Varies with the random chromosomal site of integration. • Can long-range regulatory elements protect transgenes from position effect?

34. Intron enhancers contribute to tissue-specific gene expression • Does the enhancer located in the second intron of the apolipoprotein B gene play a role in gene regulation? • What do the results suggest? • Why do you think a reporter gene was used in the experiment?

35. MARs promote formation of independent loop domains • Experiment to test the importance of MARs in transcriptional regulation of the whey acidic protein (WAP) gene. • Analyzed by Southern blot (DNA) and Northern blot (RNA). • What do the results suggest?

36. Is there a nuclear matrix? • The nuclear matrix is operationally defined as “a branched meshwork of insoluble filamentous proteins within the nucleus that remains after digestion with high salt, nucleases, and detergent.” • What forms the branching filaments remains unknown.

37. What does the nuclear matrix do? • Proposed to serve as a structural organizer within the cell nucleus. • Interaction of MARs with the nucleus is proposed to organize chromatin into loop domains and maintain chromosomal territories. • Active genes are found associated with the nuclear matrix only in cell types in which they are expressed.

38. What are the components of the nuclear matrix? • >200 types of proteins associated with the nuclear matrix. • What forms the branching filaments remains unknown. • General components include the heterogeneous nuclear ribonucleoprotein (hnRNP) complex proteins and the nuclear lamins.

39. What are the components of the nuclear matrix? • The nuclear lamina is a protein meshwork underlying the nuclear membrane. • Composed of the intermediate filament proteins lamins A, B, and C. • Internal lamins form a “veil” that branches throughout the interior of the nucleus.

40. Hutchinson-Gilford progeria syndrome • A premature aging syndrome. • Splicing mutation in the lamin A gene. • Patient cells have altered nuclear sizes and shapes, disrupted nuclear membranes, and extruded chromatin.

41. Is there a nuclear matrix? • Established by nuclear functions? • Present as a structural framework which then promotes functions?

42. 11.4 The general transcriptional machinery

43. General, but diverse, components of large multi-protein RNA polymerase machines required for promoter recognition and the catalysis of RNA synthesis.

44. Three major classes of proteins that regulate transcription • The general (basal) transcription machinery • Transcription factors • Transcriptional coactivators and corepressors

45. Components of the general transcription machinery • RNA polymerase II • General transcription factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH • Mediator

46. Four major steps of transcription initiation • Preinitiation complex assembly • Initiation • Promoter clearance and elongation • Reinitiation

47. Structure of RNA polymerase II • A 12 subunit polymerase capable of synthesizing RNA and proofreading nascent transcript.

48. Crystal structure for Saccharomyces cerevisiae RNA polymerase II • 12 subunits total (Rpb1 to 12). • 10 subunit catalytic core. • Heterodimer of Rpb4 and Rpb7. • Unstructured C-terminal domain (CTD) of Rpb1 is not seen by X-ray crystallography.

49. RNA polymerase II catalytic core • The wall prevents straight passage of nucleic acids through the cleft. • The RNA-DNA hybrid is nearly 90 to that of the entering DNA duplex. • A pore beneath the active site widens towards the outside like a funnel and includes two Mg2+ binding sites.

50. Positively charge “cleft” occupied by nucleic acids. • One side of cleft is formed by a massive, mobile “clamp.” • The active site is formed between the clamp, a “bridge helix” and a “wall”.