RNA Synthesis and Processing Transcription Transcriptional Regulation RNA Processing

1. RNA Synthesis and ProcessingTranscriptionTranscriptional RegulationRNA Processing

2. RNA Polymerase and Transcription • RNA polymerase is the principal enzyme responsible for RNA synthesis. • Like DNA polymerase, RNA polymerase is a complex enzyme made up of multiple polypeptide chains. Figure 7.1 E. coli RNA polymerase

3. Transcription in Prokaryotes • A promoter is the DNA sequence to which RNA polymerase binds to initiate transcription of a gene. Figure 7.2 Sequences of E. Coli promoters

4. 7.3 DNA footprinting • DNA Footprinting experiments identify sites at which RNA polymerase binds to promoters. • Methods that employ chemical reagents to modify and cleave DNA at particular nucleotides can be used to identify the specific DNA bases that are in contact with protein

5. Eukaryotic Transcription and RNA Polymerases • Eukaryotic cells contain three distinct nuclear RNA polymerases that transcribe different classes of genes. • The nuclear RNA polymerases recognize different promoters and transcribe distinct classes of genes. • Transcription in eukaryotes takes place on chromatin rather than on free DNA.

6. Transcription factors are specific proteins that are required for RNA polymerase II to initiate transcription. The TATA box is a regulatory DNA sequence found in the promoters of many eukaryotic genes. The TATA-binding protein, or TBP, is a basal transcription factor that binds directly to the TATA box. TBP-associated factors, or TAFs, are polypeptides associated with TBP in the general transcription factor, TFIID. General Transcription Factors

7. Initiation of Transcription by RNA Polymerase II Figure 7.12 Formation of a polymerase II transcription initiation complex

8. 7.12 Formation of a polymerase II transcription initiation complex (Part 2) • Following recruitment of RNA polymerase II to the promoter, the binding of two additional factors—TFIIE and TFIIH—completes formation of the initiation complex.

9. 7.14 RNA polymerase II/Mediator complexes • The Mediator is a large protein complex that stimulates basal transcription; it also plays a key role in linking the general transcription factors to the gene-specific transcription factors that regulate gene expression.

10. RNA polymerase I is devoted solely to the transcription of ribosomal RNA genes, which are present in tandem repeats. The promoters of ribosomal RNA genes span about 150 base pairs just upstream of the transcription initiation site. The genes for tRNAs, 5S rRNA, and some of the small RNAs involved in splicing and protein transport are transcribed by RNA Polymerase III. Transcription by RNA Polymerases I and II Figure 7.15 The ribosomal RNA gene

11. An important difference between transcriptional regulation in prokaryotes and eukaryotes results from the packaging of eukaryotic DNA into chromatin, which limits its availability as a template for transcription. Noncoding RNAs, as well as proteins, regulate transcription in eukaryotic cells via modifications in chromatin structure. Regulation of Transcription in Eukaryotes Figure 7.18 Identification of eukaryotic regulatory sequences

12. Certain cis-acting sequences regulate the expression of eukaryotic genes. Genes transcribed by RNA polymerase II have core promoter elements, including the TATA box and the Inr sequence, that serve as specific binding sites for general transcription factors. cis-Acting Regulatory Sequences: Promoters and Enhancers Eukaryotic Promoter

13. 7.20 The SV40 enhancer • Enhancers are transcriptional regulatory sequences that can be located at a site distant from the promoter. • The activity of enhancers depends on neither their distance nor their orientation with respect to the transcription initiation site.

14. 7.21 Action of enhancers

15. 7.22 DNA looping • DNA looping allows a transcription factor bound to a distant enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter • Enhancers may contain multiple sequence elements that bind different transcriptional factor.

16. An electrophoretic-mobility shift assay is a process in which a radiolabeled DNA fragment is incubated with a protein preparation and then subjected to electrophoresis through a non-denaturing gel. The binding sites of most transcription factors consist of short DNA sequences, typically spanning 6–10 base pairs. Transcription Factor Binding Sites Figure 7.24 Electrophoretic-mobility shift assay

17. Chromatin immunoprecipitation is a method for determining regions of DNA that bind transcription factors within a cell. Transcription Factor Binding Sites

18. Transcriptional activators bind to regulatory DNA sequences and stimulate transcription. Many different transcription factors contain many distinct types of DNA-binding domains. Structure and Function of Transcriptional Activators Figure 7.28 Structure of transcriptional activators

19. 7.29 Examples of DNA-binding domains

20. The activation domains of transcription factors are not as well characterized as their DNA-binding domains. Coactivators stimulate transcription by modifying chromatin structure. Structure and Function of Transcriptional Activators Figure 7.31 Action of transcriptional activators

21. Many active repressors have been found to play key roles in the regulation of transcription in animals cells. Corepressors act by modifying chromatin structure. Eukaryotic Repressors Figure 7.32 Action of eukaryotic repressors

22. Histone acetylation is the modification of histones by the addition of acetyl groups to specific lysine residues. Like acetylation, certain modifications occur at specific amino acid residues in the histone tails. Relationship of Chromatin Structure to Transcription Figure 7.34 Histone Acetylation

23. 7.34 Histone acetylation (Part 2)

24. Nucleosome remodeling factors are protein complexes that alter the arrangement or structure of nucleosomes without removing or covalently modifying the histones. Relationship of Chromatin Structure to Transcription Figure 7.36 Nucleosome remodeling factors

25. Differences in methylation are maintained following DNA replication by an enzyme that specifically methylates CpG sequences of a daughter strand that is hydorgen-bonded to a methylated parental strand. DNA Methylation Figure 7.41 Maintenance of methylation patterns

26. Processing of mRNA in Eukaryotes • Primary transcripts of eukaryotic mRNAs undergo extensive modifications before they can serve as templates for protein synthesis. • Pre-mRNA is the primary transcript that is processed to form messenger RNA in eukaryotic cells. • A 7-methylguanosine cap is what is added during the modification of the 5¢ end of a transcript. Figure 7.44 Processing of eukaryotic messenger RNAs

27. 7.44 Processing of eukaryotic messenger RNAs (Part 2)

28. Processing of mRNA in Eukaryotes A poly-A tail is a tract of about 200 adenine nucleotides added to the 3¢ ends of eukaryotic mRNAs. Polyadenylation is the process of adding a poly-A tail to a pre-mRNA. Figure 7.45 Formation of the 3’ ends of eukaryotic mRNAs

29. 7.47 Splicing of pre-mRNA • In vitro splicing reactions suggest that splicing occurs in 2 steps. • Splicing of pre-mRNA is carried out by large complexes called the spliceosomes.

30. Splicing Mechanisms Small nuclear RNAs, or snRNAs, are nuclear RNAs that range in size from 50 to 200 bases. Small nuclear ribonucleoprotein particles, or snRNPs, are complexes of snRNAs with proteins that play central roles in the splicing process.

31. Splicing Mechanisms Figure 7.48 Assembly of the spliceosome

32. 7.49 Binding of U1 snRNA to the 5’ splice site • The SR splicing factors bind to specific sequences within exons and act to recruit U1 snRNPs to the 5¢ splice site.

33. 7.50 Self-splicing introns

34. 7.51 Role of splicing factors in spliceosome assembly

35. Alternative Splicing Alternative splicing is the generation of different mRNAs by varying the pattern of pre-mRNA splicing. In the sex determination of Drosophila, alternative splicing of the same pre-mRNA determines whether a fly is male or female.

36. RNA Degradation Most of the sequences transcribed into pre-mRNA are degraded within the nucleus. Nonsense-mediated mRNA decay is a quality-control system that leads to the degradation of mRNAs that lack complete open-reading frames. Figure 7.55 Regulation of transferrin receptor mRNA stability