CHAPTER 11

1. CHAPTER 11 Gene Expression: From Transcription to Translation

2. 11.1 The Relationship between Genes and Proteins (1) • Genes store information for producing all cellular proteins. • Early observation suggested a direct relationship between genes and proteins. • Garrod studied the relationship between a specific gene, a specific enzyme, and a metabolic condition (alcaptonuria). • Beadle and Tatum formulated the “one gene–one enzyme” hypothesis.

3. The Beadle-Tatum experiment

4. The Relationship between Genes and Proteins (2) • Beadle and Tatum’s hypothesis was alter modified to “one gene–one polypeptide chain” • Mutation in a single gene causes a single substitution in an amino acid sequence of a single protein.

5. The Relationship between Genes and Proteins (3) • An Overview of the Flow of Information through the Cell • Messenger RNA (mRNA) is an intermediate between a gene and a polypeptide. • Transcription is the process by which RNA is formed from a DNA template. • Translation is the process by which proteins are synthesized in the cytoplasm from an mRNA template.

6. Overview of the flow ofinformation in eukaryotes

7. The Relationship between Genes and Proteins (4) • There are three classes of RNA in a cell: mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA). • rRNA recognizes other molecules, provide structural support, and catalyzes the chemical reaction in which amino acids are linked to one another. • tRNAs are required to translate information in the mRNA code into amino acids.

8. Structure of a bacterial ribosomal RNA

9. 11.2 An Overview of Transcription andTranslation in Both Prokaryoticand Eukaryotic Cells (1) • DNA-dependent RNA polymerases (or RNA polymerases) are responsible for transcription in both prokaryotes and eukaryotes. • These enzymes incorporate nucleotides into a strand of RNA from a DNA template. • The promoter is where the enzyme binds prior to initiating transcription. • The enzyme require the help of transcription factors to recognize the promoter.

10. Chain elongation during transcription

11. Chain elongation during transcription

12. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (2) • The newly synthesized RNA chain grows in a 5’ to 3’ direction antiparallel to the DNA. • RNA polymerase must be processive – remain attached to DNA over long stretches. • RNA polymerase must be able to move from nucleotide to nucleotide. • Nucleotides enter the polymerization reaction as trinucleotide precursors. • The reaction is driven forward by the hydrolysis of a pyrophosphate: PPi 2Pi

13. Experimental techniques to follow the activities of RNA polymerase

14. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (3) • Once polymerase has finished adding nucleotides, the DNA-RNA hybrid dissociates and the DNA double helix reforms. • There are two enzymatic activities of RNA polymerase: digestion of incorrect nucleotides and polymerization.

15. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (4) • Transcription in Bacteria • There is only one type of RNA polymerase in prokaryotes: five subunits associated to form a core enzyme. • Transcription-competent cells also have a sigma factor attached to the RNA polymerase before attaching to DNA.

16. Initiation of trancriptionin bacteria

17. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (5) • Bacterial promoters are located upstream from the site of initiation. • Two conserved regions: –35 element (consensus sequence) and Pribnow box. • Differences in the DNA sequences at both –35 element and the Pribnow box may regulate gene expression. • Termination in bacteria can either require a rho factor protein or may reach a terminator sequence without rho.

18. The basic element of a promoterregion in bacteria

19. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (6) • Transcription and Processing in Eukaryotic Cells • There are three types of RNA polymerases in eukaryotes. • Most rRNAs are transcribed by RNA polymerase I. • mRNAs are transcribed by RNA polymerase II. • tRNAs are transcribed by RNA polymerase III.

20. Eukaryotic Nuclear RNA Polymerases

21. A comparison of prokaryotic and eukaryotic RNA polymerase structure

22. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (7) • Transcription factors regulate the activity of RNA polymerases. • Newly transcribed RNAs are processed. • A primary transcript (or pre-RNA) is the initial RNA molecule synthesized. • A transcription unit is the DNA segment corresponding to a primary transcript. • A variety of small RNAs are required for RNA processing.

23. 11.3 Synthesis and Processing of Ribosomal and Transfer RNAs (1) • A eukaryotic cell may contain millions of ribosomes. • The DNA sequence encoding rRNA are called rDNA, and is typically clustered in the genome. • In nondividing cells, rDNA are clustered in the nucleoli, where ribosomes are produced.

24. The composition of a mammalian ribosome

25. The nucleolus

26. Synthesis and Processing of Ribosomal and Transfer RNAs (2) • Synthesizing the rRNA Precursor • rRNA genes are arranged in tandem. • rRNA transcription has a “Christmas tree” pattern. • Proteins that convert rRNA precursors into mature rRNA become associated with pre-rRNA during transcription. • The nonstranscribed spacer separates transcription units in a ribosomal gene cluster.

27. The synthesis of rRNA

28. The synthesis of rRNA

29. The rRNA transcription unit

30. Synthesis and Processing of Ribosomal and Transfer RNAs (3) • Processing of the rRNA Precursor • A single primary transcript (pre-rRNA) can be spliced into three rRNAs: 28S, 18S, and 5.8S. • Pre-rRNA contains large numbers of methylated nucleotides and pseudouridine residues. • Unaltered sections of the pre-rRNA are discarded.

31. Kinetic analysis of rRNA synthesisand processing

32. Proposed scheme for the processing of mammalian rRNA

33. Synthesis and Processing of Ribosomal and Transfer RNAs (4) • The Role of snoRNAs • Processing of pre-rRNA is helped by small, nucleolar RNAs (snoRNAs). • snoRNAs are packaged with proteins into snoRNPs (small, nucleolar ribonucleoproteins). • snoRNAs modify bases in pre-RNAs.

34. Modifying the pre-rRNA

35. Synthesis and Processing of Ribosomal and Transfer RNAs (5) • Synthesis and Processing of the 5S rRNA • The 5S rRNA genes are located outside the nucleolus. • It is transcribed by RNS polymerase III, which uses an internal promoter.

36. Synthesis and Processing of Ribosomal and Transfer RNAs (6) • Transfer RNAs • tRNA genes are located in small clusters scattered around the genome. • tRNAs have promoter sequences within the coding region of the gene. • During processing, the tRNA precursor is trimmed and numerous bases must be modified.

37. The arrangement of genes that code for tRNAs

38. 11.4 Synthesis and Processing of Messenger RNAs (1) • The precursors of mRNAs are represented by diverse RNAs called heterogeneous nuclearRNAs (hnRNAs). • hnRNAS are found only in the nucleus. • hnRNAs have large molecular weights. • hnRNAs are degraded after a very short time.

39. The formation of hnRNA and its conversioninto smaller mRNAs

40. Synthesis and Processing of Messenger RNAs (2) • The Machinery for mRNA Transcription • RNA polymerase II is assisted by general transcription factors (GTFs) to form the preinitiation complex (PIC). • The critical portion of the promoter lies 24-32 bases upstream from the initiation site, and contains the TATA box. • The preinitiation complex of GTFs and polymerase assemble at the TATA box.

41. Initiation of transcription from a eukaryotic polymerase II promoter

42. Initiation of transcription from a eukaryotic polymerase II promoter

43. Synthesis and Processing of Messenger RNAs (3) • The preinitiation complex assembly starts with the binding of the TATA-binding protein (TBP) to the promoter. • TBP is a subunit of the TFIID and when it binds to the promoter causes a conformation change in DNA.

44. Structural models of the formation of the preinitiation complex

45. Synthesis and Processing of Messenger RNAs (4) • Binding of TFIID sets the stage for the assembly of the complete PIC. • The three GTFs bound to the promoter allows the binding of RNA polymerase with its TFIIF. • As long as TFIID remains bound to the promoter, additional RNA polymerases may be able to attach for additional rounds of transcription.

46. Initiation of transcription by RNA polymerase II

47. Synthesis and Processing of Messenger RNAs (5) • RNA polymerase is heavily phosphorylated at the carboxyl-terminal domain (CTD). • CTD phosphorylation can be catalyzed by different protein kinases. • TFIIH acts as the protein kinase. • Termination of transcription is not well understood.

48. Synthesis and Processing of Messenger RNAs (6) • The Structure of mRNAs: Messenger RNAs share certain properties • They each code for a specific polypeptide. • They are found in the cytoplasm. • They are attached to ribosomes when translated. • Most have a noncoding segment. • Eukaryotic mRNAs modifications at their 5’ (guanosone cap) and a 3’ poly(A) tail.

49. Structure of the human -globin mRNA

50. Synthesis and Processing of Messenger RNAs (7) • Split Genes: An Unexpected Finding • The difference between hnRNA and mRNA provided early clues about RNA processing. • Eukaryotic genes contain intervening sequences which are missing from mature mRNAs. • The presence of genes with intervening sequences are called split genes.