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 ; Chap 8, pp (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 ; 263-4; Chap 8, pp (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
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
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
snRNP
3’ splice site
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
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 GA Frame 2-A GUC UAG GCA CUG A Frame 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 Eukaryotes Large=60S Small=40S Prokaryotes Large=50S
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

51. 28S
Fig. 7-28
5.8S
Ribosomes made of rRNA and protein
18S 5S
small subunit
large subunit
Come together during translation

52. Ribosomes are protein synthesis factories [Fig. 7-29]

53. Ribosomes can be free in the cytosol or attached to the ER membranes

54. Fig. 7-35: polyribosomes or polysomes

55. Fig. 7-32: Initiation of translation
Initiator rRNA-Met
+small subunit
Start codon
Large subunit binds

56. Fig. 7-32: translation
Initiator tRNA in P site
Aminoacyl-tRNA binds to A site
Ribosome shift: one codon
tRNA1 moves from P site to E site; tRNA2 moves from A site to P site
Peptide bond forms

57. Fig. 7-30:
Elongation of polypeptide
Bind
Bind: aminoacyl-tRNA anticodon to mRNA codon
Bond
Bond: make peptide bond
Shift: move ribosome along mRNA, move tRNAs
Shift

58. Stop codon at A site
Fig. 7-34: termination

59. Fig. 7-34:termination
Release factor binds
stop codon at A site
Release ribosome from mRNA
Release C terminal
from tRNA+shift

60. Protein destruction
Proteolysis: digestion of proteins by hydrolyzing peptide bonds
Proteases: enzymes that digest proteins
Many proteases aggregate to form a proteasome in eukaryotes
Proteins are tagged for destruction by a molecule called ubiquitin
Several proteins are also degraded in lysosomes in eukaryotes

61. Proteasome-mediated degradation of short-lived and unwanted proteins [Fig. 7-36]

62. Mechanism of ubiquitin-dependent
protein degradation [Becker et al. The World of the Cell, Fig ]

63. SUMMARY
Transcription
RNA processing
Translation
Protein destruction

64. Transcription of two genes seen be EM [Fig. 7-8]
Where are Polymerases?
Where are transcription start/stop sites?
Where are the 3’ and 5’ ends of the transcript?
Which direction are the RNA polymerases moving?
Why are the RNA transcripts so much shorter than the length of the DNA that encodes them?