CHAPTER 6 HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN

1. CHAPTER 6 HOW CELLS READ THE GENOME: FROM DNA TO PROTEIN • Transcription - FROM DNA TO RNA • Translation - FROM RNA TO PROTEIN • Folding and Assembly – From Unstructured to structured • RNA as a multifunctional precursor

2. Transcription Rules • Portions of DNA Sequence Are Transcribed into RNA • Transcription Produces RNA Complementary to One Strand of DNA • Cells Produce Several Types of RNA • Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop • Transcription Start and Stop Signals Are Heterogeneous in Nucleotide Sequence

3. Only Certain Portions of DNA Sequence Are Transcribed into RNA

4. Transcription Produces RNA Complementary to One Strand of DNA

5. Macromolecule levels are determined by synthesis and degradation rates RNA + Frequency of mRNA initiation + Rate of elongation + Rate of mRNA processing Rate of mRNA degradation Protein + Translation initiation, synthesis, folding, assembly Degradation

6. Stable RNAs fold into unique 3D structures

7. Bacterial RNA Polymerase Structure

8. rRNA genes are transcribed from Strong Promoters

9. Bacterial transcription cycle 1 Closed complex 2 Open complex 3 Initiation 4 Sigma release 5 Promoter clearance 6 Elongation 7 RNA Hairpin Terminator 8 Dissociation

10. E. coli s70 Promoter Consensus Sequence Very few promoters are “perfect” Consensus promoters have high initiation frequency Non-consensus promoters can be activated by accessory proteins

11. Rules of Transcription Polymerization is always 5’ to 3’ Template strand is read 3’ to 5’ Non template strand (AKA “coding”, “sense”) has same sequence as RNA transcript (except T/U changes)

12. Bacterial and eukaryotic polymerases share a common core

13. Transcription Initiation • Transcription Initiation in Eucaryotes Requires Many Proteins • RNA Polymerase II Requires General Transcription Factors • Polymerase II Also Requires Activator, Mediator, and Chromatin-modifying Proteins

14. RNA Polymerase II and General Transcription Factors- In vitro assembly order

15. GTF consensus binding sequencesTBP - TATA complexDNA distortion through minor groove interactions

16. Eukaryotic transcription initiation requires multiple levels of protein-protein interactions

17. Additional eucaryoticpost-transcriptional complexity

18. Prokaryotic Polycistronic Message, Eucaryotic Monocistronic Processed Message

19. mRNA processing factors associate with the RNA Pol II CTD

20. Splicing – elimination of noncoding (alternate) sequences • RNA Splicing Removes Intron Sequences from Newly Transcribed Pre-mRNAs • Nucleotide Sequences Signal Where Splicing Occurs • RNA Splicing Is Performed by the Spliceosome • The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements • Ordering Influences in the Pre-mRNA Help to Explain How the Proper Splice Sites Are Chosen • RNA Splicing Shows Remarkable Plasticity • Spliceosome-catalyzed RNA Splicing Probably Evolved from Self-splicing Mechanisms

21. Intron sizes and distributions vary widely

22. Splicing RNA reactants, intermediates and products

23. Alternative splicing generates different proteins in different tissues

24. Cis acting sequences

25. Splicing Mechanism

26. RNA basepairing aligns components of the Spliceosome

27. Splicing is not perfect

28. Nuclear pre-mRNA is coated with proteins

29. Splice site evolution

30. Some RNAs can splice themselves independent of any proteins

31. Poly A 3’ “tail” addition

32. Transport • Mature Eucaryotic mRNAs Are Selectively Exported from the Nucleus • Many Noncoding RNAs Are Also Synthesized and Processed in the Nucleus • The Nucleolus Is a Ribosome-Producing Factory • The Nucleus Contains a Variety of Subnuclear Structures

33. Transport of mRNA - Exchange of proteins

34. Overview of Nuclear processes Proteins are synthesized in the cytoplasm* RNA in the nucleus Ribosome assembly begins in the nucleolus

35. FROM RNA TO PROTEIN • tRNAs translate the genetic code • Mechanism of translation • Initation and termination • Protein folding and assembly • Protein degradation

36. tRNAs translate the genetic code • An mRNA Sequence Is Decoded in Sets of Three Nucleotides • tRNA Molecules Match Amino Acids to Codons in mRNA • tRNAs Are Covalently Modified Before They Exit from the Nucleus • Specific Enzymes Couple Each Amino Acid to Its Appropriate tRNA Molecule • Editing by RNA Synthetases Ensures Accuracy

37. The genetic code organized by amino acids

38. tRNA Structure

39. tRNA synthetases aminoacylate tRNAs

40. Fidelity of aminoacylation is enhanced by tRNA Synthetase editing

41. Mechanism of translation • Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain • The RNA Message Is Decoded on Ribosomes • Elongation Factors Drive Translation Forward • The Ribosome Is a Ribozyme

42. Peptide synthesis chemistry

43. Ribosome Structure

44. tRNA ratcheting on the ribosome

45. GTP hydrolysis by elongation factors drives translation and improves accuracy

46. Initation and termination • Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis • Stop Codons Mark the End of Translation • Proteins Are Made on Polyribosomes • Quality-Control Mechanisms Operate at Many Stages of Translation • There Are Minor Variations in the Standard Genetic Code • Many Inhibitors of Procaryotic Protein Synthesis Are Useful as Antibiotics

47. Translation initiation

48. Translation termination

49. Cap - PolyA interactions enhance translation efficiency

50. tmRNA mechanism of Ribosome Rescue