1. Chapter 8 Gene Expression
The Flow of Genetic Information from DNA via RNA to Protein

2. Outline of Chapter 8 The genetic code Transcription Translation
How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a sequence of amino acids
Transcription
How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a gene’s DNA template
Translation
How base pairing between mRNA and tRNAs directs the assembly of a polypeptide on the ribosome
Significant differences in gene expression between prokaryotes and eukaryotes
How mutations affect gene information and expression

3. The triplet codon represents each amino acid
20 amino acids encoded for by 4 nucleotides
By deduction:
1 nucleotide/amino acid = 41 = 4 triplet combinations
2 nucleotides/amino acid = 42 = 16 triplet combinations
3 nucleotides/amino acid = 43 = 64 triplet combinations
Must be at least triplet combinations that code for amino acids

4. The Genetic Code: 61 triplet codons represent 20 amino acids; 3 triplet codons signify stop
Figure 8.3
Fig. 8.3

5. A gene’s nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide
Charles Yanofsky – E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions
Trp- mutants in trpA
Fine structure recombination map
Determined amino acid sequences of mutants

6. Figure 8.4
Fig. 8.4

7. A codon is composed of more than one nucleotide
Different point mutations may affect same amino acid
Codon contains more than one nucleotide
Each nucleotide is part of only a single codon
Each point mutation altered only one amino acid

8. A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame studies of frameshift mutations in bacteriophage T4 rIIB gene
Figure 8.5
Fig. 8.5

9. Most amino acids are specified by more than one codon
Phenotypic effect of frameshifts depends on if reading frame is restored
Figure 8.6
Fig. 8.6

10. Cracking the code: biochemical manipulations revealed which codons represent which amino acids
The discovery of messenger RNAs, molecules for transporting genetic information
Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids
RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm

11. Polytetranucleotides Read amino acid sequence and deduced codons
Synthetic mRNAs and in vitro translation determines which codons designate which amino acids
1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated to polypeptides in vitro
Polymononucleotides
Polydinucleotides
Polytrinucleotides
Polytetranucleotides
Read amino acid sequence and deduced codons
Figure 8.7
Fig. 8.7

12. Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes
Figure 8.8
Fig. 8.8

13. 5’ to 3’ direction of mRNA corresponds to N-terminal-to-C-terminal direction of polypeptide
One strand of DNA is a template
The other is an RNA-like strand
Nonsense codons cause termination of a polypeptide chain – UAA (ocher), UAG (amber), and UGA (opal)
Figure 8.9
Fig. 8.9

14. Summary
Codon consist of a triplet codon each of which specifies an amino acid
Code shows a 5’ to 3’ direction
Codons are nonoverlapping
Code includes three stop codons, UAA, UAG, and UGA that terminate translation
Code is degenerate
Fixed starting point establishes a reading frame
UAG in an initiation codon which specifies reading frame
5’- 3’ direction of mRNA corresponds with N-terminus to C-terminus of polypeptide
Mutation modify message encoded in sequence
Frameshift mutaitons change reading frame
Missense mutations change codon of amino acid to another amino acid
Nonsense mutations change a codon for an amino acid to a stop codon

15. Do living cells construct polypeptides according to same rules as in vitro experiments?
Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene
Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code
Figure 8.10 a
Fig a

16. Proflavin treatment generates Trp- mutants
Further treatment generates Trp+ revertants
Single base insertion (Trp-) and a deletion causes reversion (Trp+)
Figure 8.10 b
Fig b

17. Genetic code is almost universal but not quite
All living organisms use same basic genetic code
Translational systems can use mRNA from another organism to generate protein
Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms

18. Transcription RNA polymerase catalyzes transcription
Promoters signal RNA polymerase where to begin transcription
RNA polymerase adds nucleotides in 5’ to 3’ direction
Terminator sequences tell RNA when to stop transcription

19. Initiation of transcription
Figure 8.11 a
Fig a

20. Elongation
Figure 8.11 b
Fig b

21. Termination
Figure 8.11 c
Fig c

22. Information flow
Figure 8.11 d
Fig d

23. Promoters of 10 different bacterial genes
Figure 8.12
Fig. 8.12

24. In eukaryotes, RNA is processed after transcription
Figure 8.13
A 5’ methylated cap and a 3’ Poly-A tail are added
Structure of the methylated cap

25. How Poly-A tail is added to 3’ end of mRNA
Figure 8.14
Fig. 8.14

26. RNA splicing removes introns
Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions)
Introns – sequences found in DNA but not in mRNA (intervening regions)
Some eukaryotic genes have many introns

27. Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns
Figure 8.15
Fig. 8.15

28. How RNA processing splices out introns and adjoins adjacent exons
Figure 8.16
Fig. 8.16

29. Splicing is catalyzed by spliceosomes
Ribozymes – RNA molecules that act as enzymes
Ensures that all splicing reactions take place in concert
Figure 8.17
Fig. 8.17

30. Alternative splicing
Different mRNAs can be produced by same transcript
Rare transplicing events combine exons from different genes
Figure 8.18
Fig. 8.18

31. Translation
Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids
tRNAs carry anticodon on one end
Three nucleotides complementary to an mRNA codon
Structure of tRNA
Primary – nucleotide sequence
Secondary – short complementary sequences pair and make clover leaf shape
Tertiary – folding into three dimensional space shape like an L
Base pairing between an mRNA codon and a tRNA anticodon directs amino acid incorporation into a growing polypeptide
Charged tRNA is covalently coupled to its amino acid

32. Secondary and tertiary structure
Figure 8.19 b
Fig b

33. Aminoacyl-tRNA syntetase catalyzes attachment of tRNAs to corresponding amino acid
Figure 8.20
Fig. 8.20

34. Base pairing between mRNA codon and tRNA anticodon determines where incorporation of amino acid occurs
Figure 8.21
Fig. 8.21

35. Wobble: Some tRNAs recognize more than one codon for amino acids they carry
Figure 8.22
Fig. 8.22

36. Rhibosomes are site of polypeptide synthesis
Ribosomes are complex structures composed of RNA and protein
Figure 8.23
Fig. 8.23

37. Mechanism of translation
Initiation sets stage for polypeptide synthesis
AUG start codon at 5’ end of mRNA
Formalmethionine (fMet) on initiation tRNA
First amino acid incorporated in bacteria
Elongation during which amino acids are added to growing polypeptide
Ribosomes move in 5’-3’ direction revealing codons
Addition of amino acids to C terminus
2-15 amino acids per second
Termination which halts polypeptide synthesis
Nonsense codon recognized at 3’ end of reading frame
Release factor proteins bind at nonsense codons and halt polypeptide synthesis

38. Initiation of translation
Figure 8.24 a
Fig a

39. Elongation
Figure 8.24 b
Fig b

40. Termination of translation
Figure 8.24 c
Fig c

41. Posttranslational processing can modify polypeptide structure
Figure 8.25
Fig. 8.25

42. Significant differences in gene expression between prokaryotes and eukaryotes
Eukaryotes, nuclear membrane prevents coupling of transcription and translation
Prokaryotic messages are polycistronic
Contain information for multiple genes
Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon
5’ untranslated leader sequence – between 5’ cap and AUG start
Only a single polypeptide produced from each gene
Initiating tRNA in prokaryotes is fMet
Initiating tRNA in eukaryotes is by unmodified Met.

43. Table 8.1

44. Table 8
Table 8.2

45. Nonsense suppression
(a) Nonsense mutation that causes incomplete nonfunctional polypeptide
(b) Nonsense-suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide
Figure 8.28
Fig. 8.28