1. CHAPTER 11 Gene Expression: From Transcription to Translation

2. 11.1 The Relationship between Genes and Proteins (1) Genes store information for producing all cellular proteins. Early observation suggested a direct relationship between genes and proteins. – Garrod studied the relationship between a specific gene, a specific enzyme, and a metabolic condition (alcaptonuria). – Beadle and Tatum formulated the “one gene–one enzyme” hypothesis.

3. The Beadle-Tatum experiment

4. The Relationship between Genes and Proteins (2) Beadle and Tatum’s hypothesis was alter modified to “one gene–one polypeptide chain” Mutation in a single gene causes a single substitution in an amino acid sequence of a single protein.

5. The Relationship between Genes and Proteins (3) An Overview of the Flow of Information through the Cell – Messenger RNA (mRNA) is an intermediate between a gene and a polypeptide. – Transcription is the process by which RNA is formed from a DNA template. – Translation is the process by which proteins are synthesized in the cytoplasm from an mRNA template.

6. Overview of the flow of information in eukaryotes

7. The Relationship between Genes and Proteins (4) There are three classes of RNA in a cell: mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA). rRNA recognizes other molecules, provide structural support, and catalyzes the chemical reaction in which amino acids are linked to one another. tRNAs are required to translate information in the mRNA code into amino acids.

8. Structure of a bacterial ribosomal RNA

9. 11.2 An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (1) DNA-dependent RNA polymerases (or RNA polymerases) are responsible for transcription in both prokaryotes and eukaryotes. These enzymes incorporate nucleotides into a strand of RNA from a DNA template. – The promoter is where the enzyme binds prior to initiating transcription. – The enzyme require the help of transcription factors to recognize the promoter.

10. Chain elongation during transcription

12. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (2) The newly synthesized RNA chain grows in a 5’ to 3’ direction antiparallel to the DNA. – RNA polymerase must be processive – remain attached to DNA over long stretches. – RNA polymerase must be able to move from nucleotide to nucleotide. Nucleotides enter the polymerization reaction as trinucleotide precursors. The reaction is driven forward by the hydrolysis of a pyrophosphate: PP i  2P i

13. Experimental techniques to follow the activities of RNA polymerase

14. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (3) Once polymerase has finished adding nucleotides, the DNA-RNA hybrid dissociates and the DNA double helix reforms. There are two enzymatic activities of RNA polymerase: digestion of incorrect nucleotides and polymerization.

15. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (4) Transcription in Bacteria – There is only one type of RNA polymerase in prokaryotes: five subunits associated to form a core enzyme. – Transcription-competent cells also have a sigma factor attached to the RNA polymerase before attaching to DNA.

16. Initiation of trancription in bacteria

17. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (5) Bacterial promoters are located upstream from the site of initiation. – Two conserved regions: –35 element (consensus sequence) and Pribnow box. Differences in the DNA sequences at both –35 element and the Pribnow box may regulate gene expression. Termination in bacteria can either require a rho factor protein or may reach a terminator sequence without rho.

18. The basic element of a promoter region in bacteria

19. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (6) Transcription and Processing in Eukaryotic Cells – There are three types of RNA polymerases in eukaryotes. Most rRNAs are transcribed by RNA polymerase I. mRNAs are transcribed by RNA polymerase II. tRNAs are transcribed by RNA polymerase III.

20. Eukaryotic Nuclear RNA Polymerases

21. A comparison of prokaryotic and eukaryotic RNA polymerase structure

22. An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (7) Transcription factors regulate the activity of RNA polymerases. Newly transcribed RNAs are processed. – A primary transcript (or pre-RNA) is the initial RNA molecule synthesized. – A transcription unit is the DNA segment corresponding to a primary transcript. – A variety of small RNAs are required for RNA processing.

23. 11.3 Synthesis and Processing of Ribosomal and Transfer RNAs (1) A eukaryotic cell may contain millions of ribosomes. The DNA sequence encoding rRNA are called rDNA, and is typically clustered in the genome. In nondividing cells, rDNA are clustered in the nucleoli, where ribosomes are produced.

24. The composition of a mammalian ribosome

25. The nucleolus

26. Synthesis and Processing of Ribosomal and Transfer RNAs (2) Synthesizing the rRNA Precursor – rRNA genes are arranged in tandem. – rRNA transcription has a “Christmas tree” pattern. – Proteins that convert rRNA precursors into mature rRNA become associated with pre-rRNA during transcription. – The nonstranscribed spacer separates transcription units in a ribosomal gene cluster.

27. The synthesis of rRNA

29. The rRNA transcription unit

30. Synthesis and Processing of Ribosomal and Transfer RNAs (3) Processing of the rRNA Precursor – A single primary transcript (pre-rRNA) can be spliced into three rRNAs: 28S, 18S, and 5.8S. – Pre-rRNA contains large numbers of methylated nucleotides and pseudouridine residues. – Unaltered sections of the pre-rRNA are discarded.

31. Kinetic analysis of rRNA synthesis and processing

32. Proposed scheme for the processing of mammalian rRNA

33. Synthesis and Processing of Ribosomal and Transfer RNAs (4) The Role of snoRNAs – Processing of pre-rRNA is helped by small, nucleolar RNAs (snoRNAs). snoRNAs are packaged with proteins into snoRNPs (small, nucleolar ribonucleoproteins). snoRNAs modify bases in pre-RNAs.

34. Modifying the pre-rRNA

35. Synthesis and Processing of Ribosomal and Transfer RNAs (5) Synthesis and Processing of the 5S rRNA – The 5S rRNA genes are located outside the nucleolus. – It is transcribed by RNS polymerase III, which uses an internal promoter.

36. Synthesis and Processing of Ribosomal and Transfer RNAs (6) Transfer RNAs – tRNA genes are located in small clusters scattered around the genome. – tRNAs have promoter sequences within the coding region of the gene. – During processing, the tRNA precursor is trimmed and numerous bases must be modified.

37. The arrangement of genes that code for tRNAs

38. 11.4 Synthesis and Processing of Messenger RNAs (1) The precursors of mRNAs are represented by diverse RNAs called heterogeneous nuclear RNAs (hnRNAs). – hnRNAS are found only in the nucleus. – hnRNAs have large molecular weights. – hnRNAs are degraded after a very short time.

39. The formation of hnRNA and its conversion into smaller mRNAs

40. Synthesis and Processing of Messenger RNAs (2) The Machinery for mRNA Transcription – RNA polymerase II is assisted by general transcription factors (GTFs) to form the preinitiation complex (PIC). – The critical portion of the promoter lies 24-32 bases upstream from the initiation site, and contains the TATA box. – The preinitiation complex of GTFs and polymerase assemble at the TATA box.

41. Initiation of transcription from a eukaryotic polymerase II promoter

43. Synthesis and Processing of Messenger RNAs (3) The preinitiation complex assembly starts with the binding of the TATA-binding protein (TBP) to the promoter. TBP is a subunit of the TFIID and when it binds to the promoter causes a conformation change in DNA.

44. Structural models of the formation of the preinitiation complex

45. Synthesis and Processing of Messenger RNAs (4) Binding of TFIID sets the stage for the assembly of the complete PIC. The three GTFs bound to the promoter allows the binding of RNA polymerase with its TFIIF. As long as TFIID remains bound to the promoter, additional RNA polymerases may be able to attach for additional rounds of transcription.

46. Initiation of transcription by RNA polymerase II

47. Synthesis and Processing of Messenger RNAs (5) RNA polymerase is heavily phosphorylated at the carboxyl-terminal domain (CTD). CTD phosphorylation can be catalyzed by different protein kinases. TFIIH acts as the protein kinase. Termination of transcription is not well understood.

48. Synthesis and Processing of Messenger RNAs (6) The Structure of mRNAs: Messenger RNAs share certain properties – They each code for a specific polypeptide. – They are found in the cytoplasm. – They are attached to ribosomes when translated. – Most have a noncoding segment. – Eukaryotic mRNAs modifications at their 5’ (guanosone cap) and a 3’ poly(A) tail.

49. Structure of the human  -globin mRNA

50. Synthesis and Processing of Messenger RNAs (7) Split Genes: An Unexpected Finding – The difference between hnRNA and mRNA provided early clues about RNA processing. – Eukaryotic genes contain intervening sequences which are missing from mature mRNAs. – The presence of genes with intervening sequences are called split genes.

51. The difference in size between hnRNAs and mRNAs

52. The discovery of intervening sequences

53. Synthesis and Processing of Messenger RNAs (8) The parts of the split gene that contribute to the mature mRNA are called exons. The intervening sequences are called introns. The discovery of genes with introns led to investigate how these genes were able to produce mRNAS lacking these sequences.

54. The discovery of introns in a eukaryotic gene

55. Synthesis and Processing of Messenger RNAs (9) Hybridization experiments supported the concept of mRNA precursors (pre-mRNAs). Loops in the DNA-RNA complex were the introns. The loops resulted from introns that were not complementary to any part of the gene.

56. Visualizing an intron in the globin gene

57. Visualizing introns in the ovalbumin gene

58. Synthesis and Processing of Messenger RNAs (10) The Processing of Eukaryotic Messenger RNAs – RNA transcripts become associated with ribonucleoproteins as they are synthesized. – During processing, a 5’ methylguanosine cap and 3’ poly(A) tails are added. – Intervening sequences are removed and exons are connected by RNA splicing.

59. Pre-mRNA transcripts are processed cotranscriptionally

60. Steps in the addition of 5’ methylguanosine cap and a 3’ poly(A) tail to a pre-mRNA

61. Synthesis and Processing of Messenger RNAs (11) RNA Splicing: Removal of Introns from a Pre- mRNA – Breaks are introduced at the 5’ and 3’ ends (splice sites). – Sequences between exon-intron boundaries are highly conserved. – Sequence most commonly found at the boundary is g/GU at the 5’ end and AG/G at the 3’ end.

62. Nucleotide sequences at the splice sites of pre-mRNAs

63. Synthesis and Processing of Messenger RNAs (12) The mechanism of RNA splicing has led to the study of RNA enzymes, or ribozymes. RNA splicing is thought to have evolved from self-splicing RNAs. An example of a self-splicing intron is the group II intron, discovered in various organisms.

64. The structure and self-splicing of group II introns

65. Synthesis and Processing of Messenger RNAs (13) The pre-mRNA is not capable of self-splicing, and requires small nuclear RNAs (snRNAs). As each hnRNA is transcribed, it becomes associated with a hnRNP. Processing occurs as each intron becomes associated with a complex called spliceosome. The spliceosome consists of small nuclear ribonucleoproteins (snRNPs).

66. Model of the assembly of the splicing machinery

67. Synthesis and Processing of Messenger RNAs (14) Removal of an intron requires: – Several snRNP particles. – Each snRNP contains a dozen or more proteins, such as the Sm protein family.

68. Synthesis and Processing of Messenger RNAs (15) snRNAs may be the catalytically active components of the snRNPs, not the proteins based on: – Pre-mRNA are catalyzed by the same pair of chemical reactions. – The snRNAs required for splicing pre-mRNA resemble group II introns. There is a proposal that the combined action of both RNA and a protein in the spliceosome catalyze the two chemical reactions required for RNA splicing.

69. Proposed structural similarity between reactions carried out by spliceosome and self-splicing

70. A mechanism for the coordination of transcription, capping, polyadenylation, and splicing.

71. Processing the ovomucoid pre-mRNA

72. Synthesis and Processing of Messenger RNAs (16) Evolutionary Implications of Split Genes and RNA Splicing – The idea of an “RNA world” suggests that RNA was the earliest molecule to both store information and catalyze reactions. – RNA splicing via spliceosomes could have evolved from self-splicing RNAs. – Exon shuffling could have played a role in the evolution of many genes.

73. Synthesis and Processing of Messenger RNAs (17) Creating New Ribozymes in the Laboratory – Ribozymes are RNA with catalytic activity. – Modified RNAs can catalyze certain reactions. – Synthesis of random RNA molecules is one of the strategies used to look for ribozymes.

74. 11.5 Small Regulatory RNAs and RNA Silencing Pathways (1) RNA interference (RNAi) results in the destruction of some mRNAs. RNAi is produced when a double-stranded RNA shares the same sequence as the target mRNA.

75. RNA interference

76. Small Regulatory RNAs and RNA Silencing Pathways (2) RNAi is part of a broader phenomenon of RNA silencing, in which small RNAs inhibit gene expression in various ways. The steps involved in RNAi include: – dsRNA is cleaved into small interfering RNAs (siRNAs) by an enzyme called Dicer. – The small dsRNAs are loaded into a complex named RISC that bind siRNA to a target RNA.

77. The formation and mechanism of action of siRNAs and miRNAs

78. Small Regulatory RNAs and RNA Silencing Pathways (3) Micro RNAs (miRNAs) – Are derived from a single-stranded precursor RNAs that contain complementary sequences that allow them to fold back to form dsRNA. – The pseudo dsRNA is cleaved to generate a pre- miRNA. – The single-stranded miRNA binds to a complementary region on an mRNA and inhibits translation of the message.

79. Small Regulatory RNAs and RNA Silencing Pathways (4) miRNAs – Are thought to play a regulatory role in development. – There are roles for miRNAs in the development of cancer.

80. Small Regulatory RNAs and RNA Silencing Pathways (5) piRNAs: A Class of Small RNAs that Function in Germ Cells – piwi-interacting RNAs (piRNAS) are small RNAs that suppress the movement of transposable elements in the germline. – There are several important differences between piRNAs and sn/miRNAs. – The mechanism by which piRNAs act to silence target RNA expression remains unclear.

81. The Human Perspective: Clinical Applications of RNA Interference (1) Strategies for using RNAi to combat disease are being developed against cancer, viruses, and some genetic disorders. Although the technique is promising, some obstacles remain: – Delivering genes for siRNAs can lead to complications. – Viral genes mutate rapidly, making some siRNAs ineffective for treatment against viral diseases.

82. The effects of siRNA on the gut epithelium of mice with induced inflammatory disease

83. 11.6 Encoding Genetic Information (1) Information stored in a gene is present in the form of a genetic code. The Properties of the Genetic Code – The codons for amino acids are non-overlapping triplets of nucleotides. – It is degenerate, some of the amino acids are specified by more than one codon.

84. The distinction between an overlapping and non-overlapping genetic code

85. Encoding Genetic Information (2) Identifying the Codons – Codon assignment was determined by transcription of artificial mRNAs. – The first two codon bases for a particular amino acid are invariant, whereas the third base may vary.

86. The genetic code

87. 11.7 Decoding the Codons: The Role of Transfer RNAs (1) The Structure of tRNAs – The amino acid is attached to the 3’ end of tRNA. – The secondary structure of tRNA resembles a cloverleaf, while the tertiary structure is an L shape. – The anticodon on tRNA complements the codon of the mRNA. – The wobble hypothesis suggests that a tRNA can recognize codons with variable third bases.

88. The structure of transfer RNAs

89. The structure of a tRNA

90. The wobble in the interaction between codon and anticodon

91. Decoding the Codons: The Role of Transfer RNAs (2) Amino Acid Activation – Specific aminoacyl-tRNA synthetases (aaRS) link amino acids with their respective tRNAs. – Energy from ATP is used to activate the amino acid, which is then transferred to the tRNA molecule. – Codons of the mRNA are interpreted according to the recognition abilities of the aaRS.

92. Structure of the interaction between a tRNA and aminoacyl-tRNA synthetase

93. 11.8 Translating Genetic Information (1) Protein synthesis (translation) is the most complex activity of the cell. Translation is divided into: – Initiation – Elongation – Termination

94. Translating Genetic Information (2) Initiation – Translation begins at the initiation codon, AUG, which then puts the ribosome in the proper reading frame. – The small ribosomal subunit identifies the correct AUG codon. – Initiation requires proteins called initiation factors or IFs (eIFs in eukaryotes).

95. Initiation of protein synthesis in bacteria

96. Translating Genetic Information (3) Initiation (continued) – In bacterial cells, the Shine-Dalgarno sequence guides the small ribosomal subunit to the correct initiation codon. – In eukaryotes, the smallest ribosomal subunit recognizes the 5’ end of the message and finds the first AUG triplet by scanning.

97. Translating Genetic Information (4) Bringing the First aa-tRNA Into the Ribosome – AUG codes for methionine so it is always the first amino acid to be incorporated into the polypeptide chain. – There are two differenet methionyl-tRNAs: one for initiation and one for the residues in the polypepide. – After the initiator tRNA is bound, the large subunit of the ribosome joins the complex.

98. Initiation of protein synthesis in eukaryotes

99. Translating Genetic Information (5) The Role of the Ribosome – Ribosomes have three sites for tRNAs: the A (aminoacyl) site, the P (peptidyl) site, and the E (exit ) site. – Ribosomes receive each tRNA in successive steps of the elongation cycle.

100. Model of the bacterial ribosome showing tRNAs bound to the A, P, and E sites of the ribosome

101. Translating Genetic Information (6) Elongation – The elongation cycle is the process of adding each subsequent amino acid to the growing polypeptide chain. With the charged amino acid in the P site, the next aminoacyl-tRNA binds to the vacant P site. Several elongation factors are required. – Peptidyl trasnferase catalyzes the peptide bond formation between amino acids.

102. Steps in the elongation in bacteria

103. Elongation in bacteria

104. Translating Genetic Information (7) Elongation (continued) – The ribosome moves three nucleotides (one codon) along the mRNA in the 5’  3’ direction during translocation. – Translocation is driven by conformational changes in an elongation factor (EF-G or eEF2). – Mutations that add or delete nucleotides that affect translocation are called frameshift mutations, and produce an abnormal sequence of amino acids.

105. Translating Genetic Information (8) Termination – Termination occurs at one of the three stop codons: UAA, UAG, or UGA; requires release factors, which recognize stop codons. – The ester bond linking the nascent polypeptide to the tRNA is hydrolyzed. – The final step is the dissociation of the mRNA from the ribosome and the disassembly of the ribosome.

106. Translating Genetic Information (9) mRNA Surveillance and Quality Control – Nonsense mutations produce stop codons and cause premature chain termination. – In some case, nonsense mutations are destroyed by nonsense-mediated decay (NMD). – NMD protects the cells from nonfunctional proteins. – The exon-junctions complex (EJC) can be used to detect an inappropriate stop.

107. Translating Genetic Information (10) Polyribosomes – A polyribosome (or polysome) is a complex of multiple ribosomes on mRNA, allowing simultaneous translation. – Polyribosomes increase the rate of protein synthesis.

108. Polyribosomes

110. Visualizing transcription and translation

111. Experimental Pathways: The Role of RNA as a Catalyst (1) RNA is capable of catalyzing a complex reaction of self-splicing. Experiments demonstrated that rRNA was able to excise the intron without additional factors.

112. Self-splicing of rRNA in Tetrahymena

113. Experimental Pathways: The Role of RNA as a Catalyst (2) A second example of RNA catalysis was shown in bacteria. The RNA portion of ribonuclease P can accurately cleave the tRNA precursor. The rRNA catalytic activity can be blocked by using antibiotics and ribonuclease.

114. Experimental evidence for catalytic activity of RNA