BIOL 200 (Section 921) Lecture # 4-5, June 22/23, 2006

1. BIOL 200 (Section 921)Lecture # 4-5, June 22/23, 2006 • UNIT 4: BIOLOGICAL INFORMATION FLOW - from DNA (gene) to RNA to protein • Reading: ECB (2nd Ed.) Chap 7, pp. 229-262; Chap 8, pp. 267-286 (focus only on parts covered in lecture). Related questions 7-8, 7-9, 7-11, 7-12, 7-14, 7-16, 7-17, 8-5a,b,d; or ECB (1st ed.) Chap 7, pp. 211-240; 263-4; Chap 8, pp. 257-270 (focus only on parts covered in lecture). Related questions 7-9, 7-10, 7-12, 7-13, 7-15, 7-18, 7-19; 8-7b,c,e.

2. Lecture # 4,5: Learning objectives • Understand the structural differences between DNA and RNA, and the directionality of transcription. • Understand the structure of a eukaryotic gene. • Describe the general mechanism of transcription, including binding, initiation, elongation and termination. Discuss factors regulating transcription. • Describe processing for all three types of RNA, and discuss why it must occur. • Describe the structural features of ribosomes. • Describe the genetic code and how it is related to the mechanisms of transcription and translation. • Describe the coupling of tRNA and discuss the specificity of amino acid tRNA synthetases. • Describe the general mechanism of translation including binding, initiation, elongation and termination.

3. Genetic information flow [Fig. 7-1] Transcription-same language (nuc. to nuc.) Translation-different languages (nuc. to protein)

4. Eucaryotic vs. Procaryotic Biol. Info Flow [Fig. 7-20]

5. DNA vs. RNA: Differences in chemistry [Fig. 7-3]

6. DNA vs RNA (Lehninger et al. Biochemistry)

7. DNA vs. RNA: Intramolecular base pairs in RNA [Fig. 7.5] and Intermolecular base pairs in DNA [Fig. 5-7] assist their folding into a 3D structure RNA DNA

9. Procaryotic vs. eucaryotic transcription [Fig. 7-12] Fig. 7-12, p. 219

10. In procaryotes, clusters of genes called operons can be carried by one mRNA [Fig. 8-6]

11. Fig. 7-9:Parts of a gene [Fig. 7-9] Transcription start site Promoter sequence terminator DNA template

12. Transcription factor binding to DNA [Fig. 8-4] What types of bonds can you identify between this DNA and protein?

13. Eukaryotic transcription factors promote RNA Polymerase II binding [Equiv. Fig. 8-10, 2nd Ed.]

15. Directions of transcription along a short portion of bacterial chromosome [Fig. 7-10, p. 218] 3’ 5’ 3’ 5’ DNA always read 3’ to 5’! RNA always made 5’ to 3’!

16. RNA Polymerase: (1) Unwinds and rewinds DNA; (2) Moves along the DNA one nucleotide at a time; (3) Catalyzes The formation of phosphodiester bonds from the energy in the Phosphate bonds of ATP, CTR, GTP and UTP.

17. Transcription by RNA polymerase in E. coli [Lehninger et al. Principles of Biochemistry]

19. introns exons Fig. 7-37 preRNA DNA in nucleus 5’ cap splicing polyA tail mature mRNA

20. Fig. 7-12: mRNAs in eukaryotes are processed Prokaryote mRNA Eukaryote mRNA 3’ tail 5’ cap

21. Fig 7-12B 5’ cap • Functions: • - Increased stability • Facilitate transport • mRNA identity

22. Bacterial vs. Eukaryotic genes [Fig. 7-13]

23. RNA splicing removes introns [Fig. 7-15] RNA splicing:(1) done by small nuclear ribonuclear protein molecules [snRNPs, called as SNURPs]and other proteins [together called SPLICEOSOME], (2) Specific nucleotide sequences are recognized at each end of the intron

24. Fig. 7-17 RNA Splicing by SnRNP+proteins=spliceosome 5’splice site 3’ splice site snRNP exon1 intron lariat formation

25. Fig. 7-17 RNA Splicing Lariat=loop of intron Spliceosome Exons joined Mature mRNA exons joined together-introns removed

26. Removal of intron in the form of a lariat [Fig. 7-16]

27. Detailed mechanism of RNA splicing [Fig. 7-17]

28. Is RNA splicing biologically important or a wasteful process? • Pre-mRNA can be spliced in different ways (alternate RNA splicing), thereby generating several different mRNAs which code for several different proteins [e.g. approx. 30,000 human genes can produce mRNAs coding for more than 100,000 proteins] • Introns quickens the evolution of new and biologically important proteins e.g. three exons of the β-globin gene code for different structural and functional regions (domains) of the polypeptide

29. Alternative splicing can produce different proteins from the same gene [Fig. 7-18]

30. Becker et al. The World of the Cell

31. Transcription of two genes seen be EM [Fig. 7-8]

32. Becker et al. The World of the Cell

33. Biological information flow in procaryotes and eukaryotes [Fig. 7-20]

34. Specialized RNA binding proteins (e.g. nuclear transport receptor) facilitate export of mature mRNA from nucleus to the cytoplasm [Fig. 7-19]

35. rRNA processing [Fig. 6-42 in Big Alberts] 45S transcript 18S 28S 5.8S 5S made elsewhere Small ribosomal subunit Large ribosomal subunit

36. Processing of transfer RNA (tRNA) [Becker et al. The World of the Cell]

37. From RNA to Protein: translation of the information • Translation: conversion of language of 4 nucleotides in mRNA into language of 20 amino acids in polypeptide • Genetic code: a set of rules (triplet code) which govern translation of the nucleotide sequence in mRNA into the amino acid sequence of a polypeptide • Codon: a group of three consecutive nucleotides in RNA which specifies one amino acid (e.g. ‘UGG’ specifies Trp and ‘AUG’ specifies Met)

38. Genetic code - triplet, universal, redundant and arbitrary [Fig. 7-21] mRNA codons Corresponding amino acid that will be attached onto tRNA

40. mRNA can be translated in three possible reading frames

41. “START” and “STOP” codons on mRNA are essential for proper mRNA translation [Becker et al. The World of the Cell, Fig. 22-6]

42. Genetic code problem:5’ AGUCUAGGCACUGA 3’ For the RNA sequence above, indicate the amino acids that are encoded by the three possible reading frames. If you were told that this segment of RNA was close to the3’ end of an mRNA that encoded a large protein, would you know which reading frame was used?

43. Frame 1-AGU CUA GGC ACU GAFrame 2-A GUC UAG GCA CUG AFrame 3-AG UCU AGG CAC UGA Frame 1-Ser – Leu – Gly - Thr

44. What are the main players involved in the process of translation • Ribosomes [“protein synthesis machines”]: carry out the process of polypeptide synthesis • tRNA [“adaptors”]: : align amino acids in the correct order along the mRNA template • Aminoacyl-tRNA synthetases: attach amino acids to their appropriate tRNA molecules • mRNA: encode the amino acid sequence for the polypeptide being synthesized • Protein factors: facilitate several steps in the translation • Amino acids: required as precursors of the polypeptide being synthesized

45. Ribosome Assembly Fig. 7-28 Eukaryotes Large=60S Small=40S Prokaryotes Large=50S Small=30S

46. Centrifugation-separates organelles and macromolecules (panel 5-4) Heavy particles move to pellet, e.g. nucleus Fixed angle Swinging bucket

47. Svedberg unit, S, shows how fast a particle sediments when subject to centrifugal force. From Becker, World of the Cell, p. 327

48. Fig. 7-23: tRNA structure Old ‘cloverleaf’ model 3-D L-shaped model Amino acid attachment H-bonds between complementary bases anticodon

49. Aminoacyl tRNA synthase charge-links tRNA with amino acids [Fig 7-26] High Energy Ester bond

50. Fig. 7-26, part 2 tRNA anticodon binds mRNA codon