Chapter 21

1. Gene Expression I: The Genetic Code and Transcription Chapter 21

2. The Genetic Code and Transcription • The coded information of DNA is used to guide RNA production and the subsequent translation into protein • The synthesis of RNA molecules is called transcription

3. The Directional Flow of Genetic Information DNA serves as a template for the synthesis of an RNA molecule which then directs the synthesis of a protein product Sometimes the RNA itself is the final product The principle of directional information flow from DNA to RNA to protein is the central dogma of molecular biology

4. Transcription and translation • Transcription refers to RNA synthesis using DNA as a template • Translation is the synthesis of protein using the information in the RNA • Messenger RNA, mRNA, is RNA that is translated into protein

5. Additional types of RNA • Ribosomal RNA, rRNA, is an integral component of the ribosome • Transfer RNA, tRNA, molecules serve as intermediaries, bringing amino acids to the ribosome • Both function during translation

6. Figure 21-1

7. Refinements of the central dogma • There are exceptions to the central dogma • For example, there are RNA viruses that carry out reverse transcription, using RNA as a template for DNA synthesis • Other viruses produce RNAs from an RNA template

8. Figure 21A-1

9. The Genetic Code • The relationship between the DNA base sequence and the linear order of amino acids in the protein products is based on a set of rules known as the genetic code • They detected a link between gene mutations and proteins

10. Mutants and metabolic pathways • Beadle and Tatum grew mutants on minimal medium with metabolic precursors of a particular amino acid or vitamin • They determined which precursors allowed the growth of each mutant • They were able to infer that each mutation disabled a single enzymatic step of a metabolic pathway, the one-gene-one-enzyme hypothesis

11. Most Genes Code for the Amino Acid Sequences of Polypeptide Chains • Linus Pauling studied the inherited disease sickle-cell anemia, in which the red blood cells assume a sickle shape • He analyzed hemoglobin using electrophoresis and found that hemoglobin of sickle cells migrated differently from normal hemoglobin • Vernon Ingram used the protease trypsin to cleave hemoglobin into fragments and then examined the peptides

12. Figure 21-2

13. Figure 21-3

14. Sickle-cell hemoglobin differs from normal hemoglobin • Ingram found just one amino acid difference between normal and sickle-cell hemoglobin • The sickle-cell hemoglobin has a valine instead of a glutamic acid; a neutral amino acid instead of a negatively charged one • This changed the one-gene-one-enzyme hypothesis; hemoglobin is not an enzyme

15. A refined hypothesis • The new hypothesis was refined to the one-gene-one-polypeptide theory: the nucleotide sequence of a gene determines the amino acid sequence of a polypeptide chain • Charles Yanofsky showed that mutations in the bacterial tryptophan synthase gene corresponded to changed amino acids in the polypeptide

16. Gene function is complicated • Most eukaryotic genes contain noncoding sequences among the coding regions of the gene • Coding sequences can be read in various combinations, each coding for a unique polypeptide chain; this is called alternative splicing • Some types of genes encode functional RNAs

17. The Genetic Code Is a Triplet Code • There are four DNA bases and 20 amino acids • A doublet code, in which two bases specify a single amino acid, is inadequate as only 16 combinations are possible • A triplet code, in which combinations of three bases specify amino acids, would have 64 possible combinations, more than enough for all 20 amino acids

18. Frameshift mutations • The gene is written in a language of three-letter words • Inserting or deleting a nucleotide causes the rest of the sequence to be read out of phase—this is a shift in the reading frame • Mutations that cause insertion or deletion of a nucleotide are thus called frameshift mutations

19. Figure 21-4

20. The Genetic Code Is Degenerate and Nonoverlapping • There are 64 combinations of nucleotide triplets and only 20 amino acids • This means the genetic code is degenerate, meaning that a particular amino acid can be specified by more than one triplet • It is also nonoverlapping; the reading frame advances three nucleotides at a time

21. Figure 21-5

22. Figure 21-5A

23. Figure 21-5B

24. The genetic code • Although the genetic code is always nonoverlapping, there are cases where a segment of DNA is translated in more than one reading frame • E.g., some viruses with very small genomes have overlapping genes, and some bacteria have genes that slightly overlap

25. Messenger RNA Guides the Synthesis of Polypeptide Chains • The genetic code refers to the order of nucleotides in the mRNA molecules that direct protein synthesis • mRNA is transcribed from DNA similarly to how DNA is replicated, but with two differences

26. Differences between mRNA synthesis and DNA replication • In mRNA synthesis, only one DNA strand is copied, called the template strand; the other strand is called the coding strand because it is similar to the mRNA sequence • In mRNA synthesis, a uracil base (U) is used instead of thymine

27. Cell-free systems • Nirenberg and Matthei pioneered the use of cell-free systems for studying protein synthesis • They decided to add synthetic RNAs of known sequence to the cell-free system • They used polynucleotide phosphorylase to make synthetic RNA molecules of predictable base composition

28. Working out the genetic code • When a single ribonucleotide is used to make RNA the RNA is called a homopolymer • When poly (U), but not other homopolymers, was added to the cell-free system, a large amount of phenylalanine was incorporated, suggesting that UUU specifies phenylalanine

29. The Codon Dictionary Was Established Using Synthetic RNA Polymers and Triplets • RNA triplets, called codons, are read by the transcriptional machinery • Further homopolymer experiments showed AAA codes for lysine, and CCC codes for proline • Copolymers were tested (containing a mixture of two nucleotides) but it was difficult to be sure which codon specified each amino acid

30. A different approach • Khorana used an approach with one important difference—he synthesized the RNA molecules in an alternating sequence • This sort of copolymer has only two codons, e.g., UAUAUAUA  UAU and AUA, and Khorana could narrow the codon assignments to either tyrosine or isoleucine • Eventually, these experiments allowed assignment of all the codons

31. Of the 64 Possible Codons in Messenger RNA, 61 Code for Amino Acids • All 64 codons are used in the translation of mRNA • 61 of them specify the addition of specific amino acids to a growing polypeptide chain • One of them, AUG, plays a role as a start codon • The remaining 3 (UAA, UAG, UGA) are stopcodons, which terminate polypeptide synthesis

32. Figure 21-6

33. The genetic code is unambiguous and degenerate • Every codon has one meaning only, the genetic code is unambiguous • It is also degenerate—many of the amino acids are specified by more than one codon • With a degenerate code, most mutations cause codon changes and a changed amino acid

34. The Genetic Code Is (Nearly) Universal Except for a few cases all organisms use the same basic genetic code In the case of mitochondria, and a few bacteria, the genetic code differs in several ways E.g., AGA is a stop codon in mammalian mitochondria and in some organisms codons specify nonstandard amino acids

35. Transcription in Bacterial Cells • The fundamental principles of transcription were first elucidated in bacteria, where molecules and mechanisms are relatively simple

36. Transcription Is Catalyzed by RNA Polymerase, Which Synthesizes RNA Using DNA as a Template • Transcription is carried out by the enzyme RNA polymerase • Bacteria have a single kind of RNA polymerase to synthesize all three classes of RNA—mRNA, tRNA, and rRNA • The RNA polymerase of E. coli has two  two  subunits, and a dissociable sigma () factor

37. Transcription Involves Four Stages: Binding, Initiation, Elongation, and Termination The DNA that gives rise to one RNA molecule is called the transcription unit Transcription begins when RNA polymerase binds to a promoter sequence (1) triggering local unwinding of the double helix RNA polymerase then initiates synthesis of RNA using one DNA strand as a template (2)

38. Figure 21-7

39. Steps of RNA synthesis (continued) • After initiation the RNA polymerase moves along the DNA template, unwinding the helix and elongating the RNA (3) • Eventually the enzyme transcribes a termination signal which stops RNA synthesis and causes release of the RNA and dissociation of the polymerase (4)

40. Binding of RNA Polymerase to a Promoter Sequence • RNA polymerase binds to a DNA promoter site, a sequence of several dozen base pairs that determines where RNA synthesis will start • The terms upstream and downstream refer to sequences located toward the 5 or 3 end of the transcription unit, respectively • The promoter is upstream of the transcribed sequence

41. Initiation of RNA Synthesis • Initiation of RNA synthesis takes place once the DNA is unwound • One of the DNA strands serves as a template for RNA synthesis, using incoming NTPs that are complementary to the template strand • RNA polymerase catalyzes the formation of a phosphodiester bond between the NTPs

42. Elongation of the RNA Chain • Chain elongation continues as RNA polymerase moves along the DNA molecule • The RNA is elongated in the 5 to 3 direction, with each new nucleotide added to the 3 end • As the polymerase moves along the DNA strand, the double helix ahead of the polymerase is unwound and the DNA behind it is rewound into a double helix

43. Figure 21-9

44. RNA polymerases have exonuclease activity • When an incorrect nucleotide is incorporated, the polymerase backs up slightly and the incorrect nucleotide and the previous one are removed • This is RNA proofreading; occasional errors in RNA molecules are not as critical as errors in DNA replication

45. Termination of RNA Synthesis • Elongation of the RNA chain proceeds until the RNA polymerase copies a sequence called the termination signal • There are two types of termination signals based on whether or not they require a protein called the rhofactor • RNA molecules that terminate without the rho factor contain a short GC-rich sequence followed by several Us

46. Types of termination signal (continued) • RNA molecules that don’t form the GC-rich hairpin require the rho factor for termination • The rho factor is an ATP-dependent unwinding enzyme moving along the RNA molecule toward the 3 end and unwinding it from the DNA template as it proceeds

47. Transcription in Eukaryotic Cells • Eukaryotic transcription involves the same four stages as prokaryotic but there are several important differences • Each of three different RNA polymerases transcribes one or more different classes of RNA • Eukaryotic promoters are more varied than bacterial ones, some are even located downstream of the gene

48. Eukaryotic transcription • Eukaryotic transcription differs from that of prokaryotes • RNA polymerases in eukaryotes require additional proteins called transcription factors, some of which must bind before the RNA polymerase can bind • Protein-protein interactions play a prominent role in eukaryotic transcription

49. Eukaryotic transcription (continued) • Eukaryotic transcription differs from that of prokaryotes • RNA cleavage is more important than termination of transcription in determining the 3 end of the transcript • Newly forming RNA molecules undergo RNA processing, chemical modification during and after transcription

50. RNA Polymerase I, II and III Carry Out Transcription in the Eukaryotic Nucleus • There are three RNA polymerases in the nucleus designated RNA polymerases I, II, and III • These differ in their location in the nucleus and the types of RNA they synthesize