RNA Synthesis and Processing

1. 7 RNA Synthesis and Processing

2. 7 RNA Synthesis and Processing • Chapter Outline • Transcription in Prokaryotes • Eukaryotic RNA Polymerases and General Transcription Factors • Regulation of Transcription in Eukaryotes • RNA Processing and Turnover

3. Introduction Regulation of gene expression allows cells to adapt to environmental changes and is responsible for the distinct activities of differentiated cell types that make up complex organisms.

4. Introduction Transcription is the first step in gene expression, and the initial level at which gene expression is regulated. RNAs in eukaryotic cells are then modified and processed in various ways.

5. Transcription in Prokaryotes Studies of E. coli have provided the model for subsequent investigations of transcription in eukaryotic cells. mRNA was discovered first in E. coli and RNA polymerase was purified and studied.

6. Transcription in Prokaryotes RNA polymerase catalyzes polymerization of ribonucleoside 5′-triphosphates (NTPs) as directed by a DNA template, always in the 5′ to 3′ direction. Transcription initiates de novo (no preformed primer required) at specific sites—this is a major step at which regulation of transcription occurs.

7. Transcription in Prokaryotes Bacterial RNA polymerase has five types of subunits. The σsubunit is weakly bound and can be separated from the others. It identifies the correct sites for transcription initiation. Most bacteria have several different σ’s that direct RNA polymerase to different start sites under different conditions.

8. Figure 7.1 E. coli RNA polymerase

9. Transcription in Prokaryotes Promoter: gene sequence to which RNA polymerase binds to initiate transcription. Promoters are 6 nucleotides long and are located at 10 and 35 base pairs upstream of the transcription start site. Consensus sequences are the bases most frequently found in different promoters.

10. Figure 7.2 Sequences of E. coli promoters

11. Transcription in Prokaryotes Experiments show the functional importance of –10 and –35 regions: Genes with promoters that differ from the consensus sequences are transcribed less efficiently. Mutations in these sequences affect promoter function. The σsubunit binds to both regions.

12. Transcription in Prokaryotes Initially, the DNA is not unwound (closed-promoter complex). The polymerase then unwinds 12–14 bases of DNA to form an open-promoter complex, allowing transcription. After addition of about ten nucleotides, σis released from the polymerase.

13. Figure 7.3 Transcription by E. coli RNA polymerase

14. Transcription in Prokaryotes During elongation, polymerase maintains an unwound region of about 15 base pairs. High-resolution structural analysis shows the βand β′ subunits form a crab-claw-like structure that grips the DNA template. A channel between these subunits contains the polymerase active site.

15. Figure 7.4 Structure of bacterial RNA polymerase

16. Transcription in Prokaryotes RNA synthesis continues until the polymerase encounters a stop signal. The most common stop signal is a symmetrical inverted repeat of a GC-rich sequence followed by seven A residues.

17. Transcription in Prokaryotes Transcription of the GC-rich inverted repeat results in a segment of RNA that can form a stable stem-loop structure. This disrupts its association with the DNA template and terminates transcription.

18. Figure 7.5 Transcription termination

19. Transcription in Prokaryotes Alternatively, transcription of some genes is terminated by a specific termination protein (Rho), which binds extended segments of single-stranded RNA.

20. Transcription in Prokaryotes Most transcriptional regulation in bacteria operates at initiation. Studies of gene regulation in the 1950s used enzymes involved in lactose metabolism. The enzymes are only expressed when lactose is present.

21. Transcription in Prokaryotes Three enzymes are involved: β-galactosidase cleaves lactose into glucose and galactose. Lactose permease transports lactose into the cell. Transacetylase inactivates toxic thiogalactosides that are transported into the cell along with lactose.

22. Figure 7.6 Metabolism of lactose

23. Transcription in Prokaryotes Genes encoding these enzymes are expressed as a single unit, called an operon. Two loci control transcription: o (operator), adjacent to transcription initiation site i (not in the operon), encodes a protein that binds to the operator.

24. Figure 7.7 Negative control of the lac operon

25. Transcription in Prokaryotes Mutants that don’t produce i gene product express the operon even when lactose is not available. This implies that the normal i gene product is a repressor, which blocks transcription when bound to o. When lactose is present in normal cells, it binds to the repressor, preventing it from binding to the operator, and the genes are expressed.

26. Transcription in Prokaryotes The lactose operon illustrates the central principle of gene regulation: Control of transcription is mediated by the interaction of regulatory proteins with specific DNA sequences.

27. Transcription in Prokaryotes Cis-acting control elements affect expression of linked genes on the same DNA molecule (e.g., the operator). Other proteins can affect expression of genes on other chromosomes (e.g., the repressor). The lac operon is an example of negative control—binding of the repressor blocks transcription.

28. Transcription in Prokaryotes Negative control: the regulatory protein (the repressor) blocks transcription. Positive control: regulatory proteins activate rather than inhibit transcription.

29. Transcription in Prokaryotes Example of positive control in E. coli: Presence of glucose (preferred energy source) represses expression of the lac operon, even if lactose is also present. This is mediated by a positive control system: If glucose decreases, levels of cAMP increase.

30. Transcription in Prokaryotes cAMP binds to the regulatory protein catabolite activator protein (CAP). This stimulates CAP to binds to its target DNA sequence upstream of the lac operon. CAP facilitates binding of RNA polymerase to the promoter.

31. Figure 7.8 Positive control of the lac operon by glucose

32. Eukaryotic RNA Polymerases and General Transcription Factors Transcription in eukaryotes: Eukaryotic cells have three RNA polymerases that transcribe different classes of genes. The RNA polymerases must interact with additional proteins to initiate and regulate transcription.

33. Eukaryotic RNA Polymerases and General Transcription Factors Transcription takes place on chromatin; regulation of chromatin structure is important in regulating gene expression.

34. Eukaryotic RNA Polymerases and General Transcription Factors Eukaryotic RNA polymerases are complex enzymes, consisting of 12 to 17 different subunits. They all have 9 conserved subunits, 5 of which are related to subunits of bacterial RNA polymerase. Yeast RNA polymerase II is strikingly similar to that of bacteria.

35. Table 7.1 Classes of genes transcribed by eukaryotic RNA polymerases

36. Figure 7.9 Structure of yeast RNA polymerase II

37. Eukaryotic RNA Polymerases and General Transcription Factors RNA polymerase II synthesizes mRNA and has been the focus of most transcription studies. Unlike prokaryotic RNA polymerase, it requires initiation factors that (in contrast to bacterial σ factors) are not associated with the polymerase.

38. Eukaryotic RNA Polymerases and General Transcription Factors General transcription factors are proteins involved in transcription from polymerase II promoters. About 10% of the genes in the human genome encode transcription factors, emphasizing the importance of these proteins.

39. Eukaryotic RNA Polymerases and General Transcription Factors Promoters contain several different sequence elements surrounding their transcription sites. The TATA box resembles the –10 sequence of bacterial promoters. Others include initiator (Inr) elements, TFIIB recognition elements (BRE), and downstream elements DCE, MTE, and DPE).

40. Figure 7.10 Formation of a polymerase II preinitiation complex in vitro (Part 1)

41. Eukaryotic RNA Polymerases and General Transcription Factors Five general transcription factors are required for initiation of transcription in vitro. General transcription factor TFIID is composed of multiple subunits, including the TATA-binding protein (TBP) and other subunits (TAFs) that bind to the Inr, DCE, MTE, and DPE sequences.

42. Eukaryotic RNA Polymerases and General Transcription Factors Several other transcription factors (TFIIB, TFIIF, TFIIE, and TFIIH) bind in association with the RNA polymerase II to form the preinitiation complex.

43. Figure 7.10 Formation of a polymerase II preinitiation complex in vitro (Part 2)

44. Eukaryotic RNA Polymerases and General Transcription Factors Within a cell, additional factors are required to initiate transcription. These include Mediator, a protein complex of more than 20 subunits; it interacts with both general transcription factors and RNA polymerase.

45. Figure 7.11 RNA polymerase II/Mediator complexes and transcription initiation

46. Eukaryotic RNA Polymerases and General Transcription Factors RNA polymerase I transcribes rRNA genes, which are present in tandem repeats. Transcription yields a large 45S pre-rRNA, which is processed to yield the 28S, 18S, and 5.8S rRNAs.

47. Figure 7.12 The ribosomal RNA gene

48. Eukaryotic RNA Polymerases and General Transcription Factors Promoters of rRNA genes are recognized by two transcription factors which recruit RNA polymerase I to form and initiation complex. UBF (upstream binding factor) SL1 (selectivity factor 1)

49. Figure 7.13 Initiation of rDNA transcription

50. Eukaryotic RNA Polymerases and General Transcription Factors Genes for tRNAs, 5S rRNA, and some of the small RNAs are transcribed by polymerase III. They are expressed from three types of promoters.