1. Chapter 12 *Lecture Outline Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. *See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes.

2. INTRODUCTION At the molecular level, a gene is a segment of DNA used to make a functional product –either an RNA or a polypeptide Transcription is the first step in gene expression 12-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. TRANSCRIPTION Transcription literally means the act or process of making a copy In genetics, the term refers to the copying of a DNA sequence into an RNA sequence The structure of DNA is not altered as a result of this process –It can continue to store information Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-3

4. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Structural genes encode the amino acid sequence of a polypeptide Transcription of a structural gene produces messenger RNA, usually called mRNA The mRNA nucleotide sequence determines the amino acid sequence of a polypeptide during translation The synthesis of functional proteins determines an organisms traits This path from gene to trait is called the central dogma of genetics Refer to Figure 12.1 Gene Expression 12-4

5. Figure 12.1 12-5 The central dogma of genetics makes DNA copies that are transmitted from cell to cell and from parent to offspring. DNA replication: produces an RNA copy of a gene. Chromosomal DNA: stores information in units called genes. Transcription: produces a polypeptide using the information in mRNA. Translation: Gene Polypeptide: becomes part of a functional protein that contributes to an organism's traits. Messenger RNA: a temporary copy of a gene that contains information to make a polypeptide. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

6. 12.1 OVERVIEW OF TRANSCRIPTION A key concept is that DNA base sequences define the beginning and end of a gene and regulate the level of RNA synthesis Another important concept is that proteins must recognize and act on DNA for transcription to occur Gene expression is the overall process by which the information within a gene is used to produce a functional product which can, in concert with environmental factors, determine a trait Figure 12.2 shows common organization of a bacterial gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-6

7. Figure 12.2 12-7 Signals the end of protein synthesis DNA: Regulatory sequences: site for the binding of regulatory proteins; the role of regulatory proteins is to influence the rate of transcription. Regulatory sequences can be found in a variety of locations. Promoter: site for RNA polymerase binding; signals the beginning of transcription. Terminator: signals the end of transcription. mRNA: Ribosome-binding site: site for ribosome binding; translation begins near this site in the mRNA. In eukaryotes, the ribosome scans the mRNA for a start codon. Start codon: specifies the first amino acid in a polypeptide sequence, usually a formylmethionine (in bacteria) or a methionine (in eukaryotes). Codons: 3-nucleotide sequences within the mRNA that specify particular amino acids. The sequence of codons within mRNA determines the sequence of amino acids within a polypeptide. Stop codon: specifies the end of polypeptide synthesis. Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides. DNA mRNA PromoterRegulatory sequence Terminator Ribosome binding site Transcription Start codon Stop codon 5′3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Many codons

8. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The DNA strand that is actually transcribed (used as the template) is termed the template strand The RNA transcript is complementary to the template strand The opposite strand is called the coding strand or the sense strand as well as the nontemplate strand The base sequence is identical to the RNA transcript Except for the substitution of uracil in RNA for thymine in DNA Transcription factors recognize the promoter and regulatory sequences to control transcription mRNA sequences such as the ribosomal-binding site and codons direct translation Gene Expression Requires Base Sequences 12-8

9. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcription occurs in three stages Initiation Elongation Termination These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA sequences The Stages of Transcription 12-9

10. DNA of a gene PromoterTerminator Completed RNA transcript RNA polymerase 5′ end of growing RNA transcript Open complex Initiation: The promoter functions as a recognition site for transcription factors (not shown). The transcription factor(s) enables RNA polymerase to bind to the promoter. Following binding, the DNA is denatured into a bubble known as the open complex. Elongation/synthesis of the RNA transcript: RNA polymerase slides along the DNA in an open complex to synthesize RNA. Termination: A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA. RNA polymerase Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12-10 Figure 12.3 Transcription

11. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Once they are made, RNA transcripts play different functional roles Refer to Table 12.1 Well over 90% of all genes are structural genes which are transcribed into mRNA Final functional products are polypeptides The other RNA molecules in Table 12.1 are never translated Final functional products are RNA molecules RNA Transcripts Have Different Functions 12-11

12. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The RNA transcripts from nonstructural genes are not translated They do have various important cellular functions They can still confer traits In some cases, the RNA transcript becomes part of a complex that contains protein subunits For example Ribosomes Spliceosomes Signal recognition particles RNA Transcripts Have Different Functions 12-12

13. 12-13

14. 12.2 TRANSCRIPTION IN BACTERIA Our molecular understanding of gene transcription came from studies involving bacteria and bacteriophages Indeed, much of our knowledge comes from studies of a single bacterium –E. coli, of course In this section we will examine the three steps of transcription as they occur in bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-14

15. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Promoters are DNA sequences that “promote” gene expression More precisely, they direct the exact location for the initiation of transcription Promoters are typically located just upstream of the site where transcription of a gene actually begins The bases in a promoter sequence are numbered in relation to the transcription start site Refer to Figure 12.4 Promoters 12-15

16. Template strand Transcription Coding strandTranscriptional start site 16 –18 bp +1 –35 sequence–10 sequence Promoter region G T C A T A C G A T A T T A T A T A T A A T A T A T 3′5′ 3′ RNA A Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-16 Figure 12.4 The conventional numbering system of promoters Bases preceding the start site are numbered in a negative direction There is no base numbered 0 Bases to the right are numbered in a positive direction Most of the promoter region is labeled with negative numbers

17. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-17 Figure 12.4 The conventional numbering system of promoters The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level Sometimes termed the Pribnow box, after its discoverer Sequence elements that play a key role in transcription Template strand Transcription Coding strandTranscriptional start site 16 –18 bp +1 –35 sequence–10 sequence Promoter region G T C A T A C G A T A T T A T A T A T A A T A T A T 3′5′ 3′ RNA A

18. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-18 Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters The most commonly occurring bases For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences of the -35 and -10 regions –35 region lac operon Consensus lacI trp operon rrn X rec A tRNA tyr +1 TTTACATATGTT A N 17 N6N6 GCGCAACATGAT A N 17 N7N7 TTGACA TTAACT A N 17 N7N7 TTGTCT TAATAT A N 16 N7N7 TTGATATATAAT A N 16 N7N7 TTCCAATATACT A N 17 N7N7 TTTACATATGAT TTGACATATAAT A N 16 N7N7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. –10 region lex A Transcribed

19. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA polymerase is the enzyme that catalyzes the synthesis of RNA In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme Five subunits =  2  ’  Sigma factor One subunit =  These subunits play distinct functional roles Initiation of Bacterial Transcription 12-19

20. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The RNA polymerase holoenzyme binds loosely to the DNA It then scans along the DNA, until it encounters a promoter region When it does, the sigma factor recognizes both the –35 and –10 regions A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA Refer to Figure 12.6 Initiation of Bacterial Transcription 12-20

21. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-21 Figure 12.6 Amino acids within the  helices hydrogen bond with bases in the -35 and -10 promoter sequences Binding of  factor protein to DNA double helix  Turn α helices binding to the major groove Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

22. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The binding of the RNA polymerase to the promoter forms the closed complex Then, the open complex is formed when the TATAAT box in the -10 region is unwound A short RNA strand is made within the open complex The sigma factor is released at this point This marks the end of initiation The core enzyme now slides down the DNA to synthesize an RNA strand This is known as the elongation phase 12-22

23. 12-23 Figure 12.7 –10 –35 –10 RNA polymerase holoenzyme After sliding along the DNA, σ factor recognizes a promoter, and RNA polymerase holoenzyme forms a closed complex. An open complex is formed, and a short RNA is made. σ factor is released, and the core enzyme is able to proceed down the DNA. σ factor RNA transcript Open complex Closed complex Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Promotor region RNA polymerase core enzyme

24. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The RNA transcript is synthesized during the elongation stage The DNA strand used as a template for RNA synthesis is termed the template or antisense strand The opposite DNA strand is called the coding strand It has the same base sequence as the RNA transcript Except that T in DNA corresponds to U in RNA Elongation in Bacterial Transcription 12-24

25. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The open complex formed by the action of RNA polymerase is about 17 bases long Behind the open complex, the DNA rewinds back into a double helix On average, the rate of RNA synthesis is about 43 nucleotides per second! Figure 12.8 depicts the key points in the synthesis of an RNA transcript Elongation in Bacterial Transcription 12-25

26. 12-26 Similar to the synthesis of DNA via DNA polymerase Figure 12.8 Key points: RNA polymerase slides along the DNA, creating an open complex as it moves. The DNA strand known as the template strand is used to make a complementary copy of RNA as an RNA–DNA hybrid. RNA polymerase moves along the template strand in a 3′ to 5′ direction, and RNA is synthesized in a 5′ to 3′ direction using nucleoside triphosphates as precursors. Pyrophosphate is released (not shown). The complementarity rule is the same as the AT/GC rule except that U is substituted for T in the RNA. 3′ 5′ 3′ 5′ RNA polymerase Direction of transcription Rewinding of DNA RNA Open complex Coding strand Template strand Unwinding of DNA Nucleotide being added to the 3′ end of the RNA RNA–DNA hybrid region Template strand C G G T T A A G C C A U Coding strand Nucleoside triphosphates Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.s

27. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Termination is the end of RNA synthesis It occurs when the short RNA-DNA hybrid of the open complex is forced to separate This releases the newly made RNA as well as the RNA polymerase E. coli has two different mechanisms for termination 1. rho-dependent termination Requires a protein known as  (rho) 2. rho-independent termination Does not require  Termination of Bacterial Transcription 12-27

28. 5′ 3′ 5′ Terminator rut RNA polymerase reaches the terminator. A stem-loop causes RNA polymerase to pause. Stem-loop Terminator RNA polymerase pauses due to its interaction with the stem-loop structure. ρ protein catches up to the open complex and separates the RNA-DNA hybrid. 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ρ recognition site (rut) ρ recognition site in RNA ρ protein binds to the rut site in RNA and moves toward the 3′ end. ρ protein 12-28  -dependent termination Figure 12.10 Rho protein is a helicase rho utilization site

29. Stem-loop that causes RNA polymerase to pause U-rich RNA in the RNA-DNA hybrid 5′ 3′ While RNA polymerase pauses, the U-rich sequence is not able to hold the RNA-DNA hybrid together. Termination occurs. NusA Terminator U U U U Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12-29  -independent termination is facilitated by two sequences in the RNA –1. A uracil-rich sequence located at the 3’ end of the RNA –2. A stem-loop structure upstream of the uracil-rich sequence  -independent termination Figure 12.11 U RNA -A DNA hydrogen bonds are relatively weak No protein is required to physically remove the RNA from the DNA This type of termination is also called intrinsic Stabilizes the RNA pol pausing

30. 12.3 TRANSCRIPTION IN EUKARYOTES Many of the basic features of gene transcription are very similar in bacteria and eukaryotes However, gene transcription in eukaryotes is more complex –Larger, more complex cells (organelles) –Added cellular complexity means more genes that encode proteins are required –Multicellularity adds another level of regulation express genes only in the correct cells at the proper time Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-30

31. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nuclear DNA is transcribed by three different RNA polymerases RNA pol I Transcribes all rRNA genes (except for the 5S rRNA) RNA pol II Transcribes all structural genes Thus, synthesizes all mRNAs Transcribes some snRNA genes RNA pol III Transcribes all tRNA genes And the 5S rRNA gene Eukaryotic RNA Polymerases 12-31

32. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display All three are very similar structurally and are composed of many subunits There is also a remarkable similarity between the bacterial RNA pol and its eukaryotic counterparts Refer to Figure 12.13 Eukaryotic RNA Polymerases 12-32

33. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (a) Structure of a bacterial RNA polymerase (b) Schematic structure of RNA polymerase Structure of a eukaryotic RNA polymerase II (yeast) Transcribed DNA (upstream) 5′ Jaw Clamp Rudder Catalytic site Wall Bridge NTPs enter through a pore Mg 2+ Entering DNA (downstream) Transcription Lid Exit 3′ 5′ © From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution." Science, Vol. 292:5523, 1863-1876, June 8, 2001. © From Seth Darst, Bacterial RNA polymerase. Current Opinion in Structural Biology. Reprinted with permission of the author. Figure 12.12 12-33 Structure of RNA polymerase

34. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Eukaryotic promoter sequences are more variable and often more complex than those of bacteria For structural genes, at least three features are found in most promoters Regulatory elements TATA box Transcriptional start site Refer to Figure 12.13 Sequences of Eukaryotic Structural Genes 12-34

35. TATA box Core promoter Transcription Transcriptional start site DNA Coding-strand sequences:TATAAA Common location for regulatory elements such as GC and CAAT boxes –100–50–25+1 Py 2 CAPy 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12-35 Usually an adenine The core promoter is relatively short –It consists of the TATA box and transcriptional start site Important in determining the precise start point for transcription The core promoter by itself produces a low level of transcription –This is termed basal transcription Figure 12.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36. 12-36 Regulatory elements are short DNA sequences that affect the binding of RNA polymerase to the promoter Transcription factors (proteins) bind to these elements and influence the rate of transcription –They are two types of regulatory elements Enhancers –Stimulate transcription Silencers –Inhibit transcription –They vary widely in their locations but are often found in the –50 to –100 region Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display TATA box Core promoter Transcription Transcriptional start site DNA Coding-strand sequences:TATAAA Common location for regulatory elements such as GC and CAAT boxes –100–50–25+1 Py 2 CAPy5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 12.13

37. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Factors that control gene expression can be divided into two types, based on their “location” cis-acting elements DNA sequences that exert their effect only over a particular gene Example: TATA box, enhancers and silencers trans-acting elements Regulatory proteins that bind to such DNA sequences Sequences of Eukaryotic Structural Genes 12-37

38. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Three categories of proteins are required for basal transcription to occur at the promoter RNA polymerase II Five different proteins called general transcription factors (GTFs) A protein complex called mediator Figure 12.14 shows the assembly of transcription factors and RNA polymerase II at the TATA box RNA Polymerase II and its Transcription Factors 12-38

39. TFIID TFIIB TFIID TFIIB TFIIDTFIIB TFIID TFIIF TATA box TFIID binds to the TATA box. TFIID is a complex of proteins that includes the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). TFIIB binds to TFIID. TFIIB acts as a bridge to bind RNA polymerase II and TFIIF. TFIIE and TFIIH bind to RNA polymerase II to form a preinitiation or closed complex. TFIIH acts as a helicase to form an open complex. TFIIH also phosphorylates the CTD domain of RNA polymerase II. CTD phosphorylation breaks the contact between TFIIB and RNA polymerase II. TFIIB, TFIIE, and TFIIH are released. RNA polymerase II Preinitiation complex Open complex CTD domain of RNA polymerase II PO 4 TFIIF TFIIE TFIID TFIIB TFIIF TFIIE TFIIH Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. PO 4 TFIIH 12-39 Figure 12.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A closed complex RNA pol II can now proceed to the elongation stage Released after the open complex is formed

40. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Basal transcription apparatus RNA pol II + the five GTFs The third component required for transcription is a large protein complex termed mediator It mediates interactions between RNA pol II and various regulatory transcription factors Its subunit composition is complex and variable Mediator may phosphorylate the CTD of RNA polymerase II and it may regulate the ability of TFIIH to phosphorylate the CTD Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation 12-40

41. 12-41

42. RNA Pol II transcriptional termination Pre-mRNAs are modified by cleavage near their 3’ end with subsequent attachment of a string of adenines Transcription terminates 500 to 2000 nucleotides downstream from the polyA signal There are two models for termination –Further research is needed to determine if either, or both are correct Refer to figure 12.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-42

43. RNA polymerase II transcribes a gene past the polyA signal sequence. The RNA is cleaved just past the polyA signal sequence. RNA polymerase continues transcribing the DNA. PolyA signal sequence 3′ 5′ 3′ Allosteric model: After passing the polyA signal sequence, RNA polymerase II is destabilized due to the release of elongation factors or the binding of termination factors (not shown). Termination occurs. Torpedo model: An exonuclease binds to the 5′ end of the RNA that is still being transcribed and degrades it in a 5′ to 3′ direction. 3′ 5′ 3′ 5′ Exonuclease catches up to RNA polymerase II and causes termination. Exonuclease 3′ 5′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-43 Figure 12.15 Possible mechanisms for Pol II termination

44. 12.4 RNA MODIFICATION Analysis of bacterial genes in the 1960s and 1970 revealed the following: –The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA –The sequence of codons in the mRNA provides the instructions for the sequence of amino acids in the polypeptide This is termed the colinearity of gene expression Analysis of eukaryotic structural genes in the late 1970s revealed that they are not always colinear with their functional mRNAs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-44

45. 12.4 RNA MODIFICATION Instead, coding sequences, called exons, are interrupted by intervening sequences or introns Transcription produces the entire gene product –Introns are later removed or excised –Exons are connected together or spliced This phenomenon is termed RNA splicing –It is a common genetic phenomenon in eukaryotes –Occurs occasionally in bacteria as well Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-45

46. 12.4 RNA MODIFICATION Aside from splicing, RNA transcripts can be modified in several ways –For example Trimming of rRNA and tRNA transcripts 5’ Capping and 3’ polyA tailing of mRNA transcripts –Refer to Table 12.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-46

47. 12-47

48. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many nonstructural genes are initially transcribed as a large RNA This large RNA transcript is enzymatically cleaved into smaller functional pieces Figure 12.16 shows the processing of mammalian ribosomal RNA Processing 12-48

49. Promoter Transcription Cleavage (the light pink regions are degraded) 45S rRNA transcript 18S5.8S28S 5′3′ 18S5.8S28S 18S5.8S28S rRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-49 Figure 12.16 Functional RNAs that are key in ribosome structure This processing occurs in the nucleolus

50. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transfer RNAs are also made as large precursors These have to be cleaved at both the 5’ and 3’ ends to produce mature, functional tRNAs Cleaved by exonuclease and endonuclease exonucleases cleave a covalent bond between two nucleotides at one end of a strand endonucleases can cleave bonds within a strand Figure 12.17 shows the trimming of a precursor tRNA Interestingly, the cleavage occurs differently at the 5’ end and the 3’ end Processing 12-50

51. 3′5′ Endonuclease Exonuclease (RNaseD) P = Pseudouridine T = 4-Thiouridine IP = 2-Isopentenyladenosine Endonuclease (RNaseP) T T A P P IP C C Anticodon Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. m G = Methylguanosine mGmG Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-51 Figure 12.17 Found to contain both RNA and protein subunits However, RNA contains the catalytic activity Therefore, it is a ribozyme Covalently modified bases

52. Experiment 12A: Identification of Introns Via Microscopy In the late 1970s, several research groups investigated the presence of introns in eukaryotic structural genes One of these groups was led by Phillip Leder –Leder used electron microscopy to identify introns in the  -globin gene It had been cloned earlier –Leder used a strategy that involved hybridization Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-52

53. Experiment 12A: Identification of Introns Via Microscopy Double-stranded DNA of the cloned  -globin gene is first denatured –Then mixed with mature  -globin mRNA The mRNA is complementary to the template strand of the DNA –So the two will bind or hybridize to each other If the DNA is allowed to renature, this complex will prevent the reformation of the DNA double helix –Refer to Figure 12.18 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-53

54. (a) No introns in the DNA + DNA region complementary to mRNA R loop mRNA Discontinuous regions of DNA that are complementary to mRNA Mature mRNA Pre-mRNA Splicing Transcription mRNA bound to DNA Template strand Coding strand Template strand mRNA bound to DNA mRNA bound to template strand R loop Intron DNA The coding strand then binds to the template strand, but it loops out where the RNA is already bound. Intron DNA of template strand Intron 55 5 3 3 5 3 5 53 3 3 53 5 5 3 3 5 3 Mix together denatured DNA and mature mRNA. The mature mRNA binds to template strand, which causes the intron DNA to loop out. (b) One intron in the DNA. The intron in the pre-mRNA is spliced out. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-54 Figure 12.18 RNA displacement loop mRNA cannot hybridize here Because the intron has been spliced out of the mRNA

55. The Hypothesis –The  -globin gene from the mouse contains one or more introns Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 12.19 12-55

56. Figure 12.19 12-56 Mix together the β-globin mRNA and cloned DNA of the β-globin gene. Isolate mature mRNA for the mouse β-globin gene. Note: Globin mRNA is abundant in reticulocytes, which are immature red blood cells. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Experimental level Conceptual level 1. 2. Separate the double-stranded DNA and allow the mRNA to hybridize. This is done using 70% formamide, at 52°C, for 16 hours. 3. Dilute the sample to decrease the formamide concentration. This allows the DNA to re-form a double-stranded structure. Note: The DNA cannot form a double-stranded structure in regions where the mRNA has already hybridized. 4. Spread the sample onto a microscopy grid. 5. Stain with uranyl acetate and shadow with heavy metal. Note: The technique of electron microscopy is described in the Appendix. 6. View the sample under the electron microscope. 7. 70% formamide Incubator Platinum electrode Platinum atoms Vacuum evaporator Specimen mRNA Solution of cloned β-globin DNA β-globin DNA Solution of β-globin mRNA Cloned β-globin DNA

57. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R loop Intron R loop Data from: Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, B. M., Sullivan, M., Maizel, J.V., and Leder, P. (1978) Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene. Proc. Natl. Acad. Sci. USA 75:725–729.  “  ”  Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-57 The Data

58. Interpreting the Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-58 Hybridization caused the formation of two R loops, separated by a double- stranded DNA region This suggests that the  -globin gene contains introns Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R loop Intron R loop Data from: Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, B. M., Sullivan, M., Maizel, J.V., and Leder, P. (1978) Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene. Proc. Natl. Acad. Sci. USA 75:725–729.  “  ” 

59. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Three different splicing mechanisms have been identified Group I intron splicing Group II intron splicing Spliceosome All three cases involve Removal of the intron RNA Linkage of the exon RNA by a phosphodiester bond Splicing 12-59

60. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Splicing among group I and II introns is termed self-splicing Splicing does not require the aid of enzymes Instead the RNA itself functions as its own ribozyme Group I and II differ in the way that the intron is removed and the exons connected Refer to Figure 12.20 Group I and II self-splicing can occur in vitro without the additional proteins However, in vivo, proteins known as maturases often enhance the rate of splicing 12-60

61. H H OH H G G G O CH 2 OH H OH H O CH 2 OH Guanosine HH O OH H CH 2 O O P P A Self-splicing introns (relatively uncommon) Exon 1 Intron Exon 2Exon 1 Intron Exon 2 Guanosine binding site G G P HH O O H CH 2 O O P P P A 3′ OH 3′ 5′3′5′ OH G G HH O O H CH 2 O O P P A RNA 5′ (a) Group I(b) Group II Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ 2′ 3′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-61 Figure 12.20 Free guanosine bound to site in intron breaks the bond between exon 1 and intron and becomes attached to 5’ end of intron 2’ hydroxyl from adenine within intron breaks bond between exon 1 and intron Results in Exon 1 and 2 covalently joined and free, linear intron Results in Exon 1 and 2 linkage and intron as lariat

62. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-62 Figure 12.20 In eukaryotes, the transcription of structural genes produces a long transcript known as pre-mRNA This RNA is altered by splicing and other modifications, before it leaves the nucleus Splicing in this case requires the aid of a multicomponent structure known as the spliceosome Intron removed via spliceosome (very common in eukaryotes) HH O OH H CH 2 O O P P A Exon 1 Intron Exon 2 Spliceosome HH O O H CH 2 O O P P P A 3′ OH 3′ HH O H CH 2 O O P P A P O 3′5′ mRNA (c) Pre-mRNA 2′ 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

63. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-63

64. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The spliceosome is a large complex that splices pre-mRNA It is composed of several subunits known as snRNPs (pronounced “snurps”) Each snRNP contains small nuclear RNA and a set of proteins Pre-mRNA Splicing 12-64

65. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The subunits of a spliceosome carry out several functions 1. Bind to an intron sequence and precisely recognize the intron-exon boundaries 2. Hold the pre-mRNA in the correct configuration 3. Catalyze the chemical reactions that remove introns and covalently link exons Pre-mRNA Splicing 12-65

66. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-66 Figure 12.21 Intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21 Sequences shown in bold are highly conserved Corresponds to the boxed adenine in Figure 12.22 Serve as recognition sites for the binding of the spliceosome The pre-mRNA splicing mechanism is shown in Figure 12.22 3′ 5′ 3′ splice siteBranch site IntronExon Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. UACUUAUCC Py 12 N Py AGG A / C GGU Pu AGUA 5′ splice site

67. U1 3′5′ 5′ splice site3′ splice site Branch site A GU Exon 1Exon 2 U1 binds to 5′ splice site. U2 binds to branch site. AG 3′ 5′ A U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together. U1 snRNPU2 snRNP 3′ 5′ A U5 snRNP U4/U6 snRNP U2 12-67 Intron loops out and exons brought closer together Figure 12.22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

68. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-68 Figure 12.22 Intron will be degraded and the snRNPs used again U1 U4 3′ 5′ 3′5′ 5′ splice site is cut. 5′ end of intron is connected to the A in the branch site to form a lariat. U1 and U4 are released. 3′ splice site is cut. Exon 1 is connected to exon 2. The intron (in the form of a lariat) is released along with U2, U5, and U6. The intron will be degraded. A A U5 U6 U5 U6 U2 Intron plus U2, U5, and U6 Two connected exons Exon 1 Exon 2 U2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cleavage may be catalyzed by snRNA molecules within U2 and U6

69. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display One benefit of genes with introns is a phenomenon called alternative splicing A pre-mRNA with multiple introns can be spliced in different ways This will generate mature mRNAs with different combinations of exons This variation in splicing can occur in different cell types or during different stages of development Intron Advantage? 12-69

70. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene This allows an organism to carry fewer genes in its genome Intron Advantage? 12-70

71. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most mature mRNAs have a 7-methylguanosine covalently attached at their 5’ end This event is known as capping Capping occurs as the pre-mRNA is being synthesized by RNA pol II Usually when the transcript is only 20 to 25 bases long As shown in Figure 12.23, capping is a three-step process Capping 12-71

72. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-72 Figure 12.23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P H H OH HH Base O CH 2 O O O PO-O- HH OH HH Base O OO O PCH 2 O–O– O P O–O– O O P O–O– Rest of mRNA H H OH HH Base O CH 2 Rest of mRNA RNA 5′-triphosphatase removes a phosphate. Guanylyltransferase hydrolyzes GTP. The GMP is attached to the 5′ end, and PP i is released. PP i PiPi 5′ 3′ OO O P O–O– O P O–O– O O P O–O– O–O– O O O O–O– O O O PO–O– OO O P O–O– O P O–O– O–O– O–O–

73. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-73 Figure 12.23 H H OH HH Base O CH 2 Rest of mRNA HH OH HH Base O CH 2 CH 3 CH 2 Rest of mRNA Methyltransferase attaches a methyl group. 7-methylguanosine cap H H H OH HO O NH 2 H N N H N O N H H H OH HO O NH 2 H N N H N O N + O O P O–O– O O O PO–O– OO O P O–O– O O P O–O– O O P O–O– O O O P O–O– OO O P O–O– O O P O–O– Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

74. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The 7-methylguanosine cap structure is recognized by cap-binding proteins Cap-binding proteins play roles in the Movement of some RNAs into the cytoplasm Early stages of translation Splicing of introns Capping 12-74

75. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most mature mRNAs have a string of adenine nucleotides at their 3’ ends This is termed the polyA tail The polyA tail is not encoded in the gene sequence It is added enzymatically after the gene is completely transcribed The attachment of the polyA tail is shown in Figure 12.24 Tailing 12-75

76. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ 5′ 3′ Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. PolyA-polymerase adds adenine nucleotides to the 3′ end. Polyadenylation signal sequence AAUAAA PolyA tail AAAAAAAAAAAA.... Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-76 Figure 12.24 Consensus sequence in higher eukaryotes Appears to be important in the stability of mRNA and the translation of the polypeptide Length varies between species From a few dozen adenines to several hundred

77. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Change in the nucleotide sequence of an RNA Can involve addition or deletion of particular bases Can also occur through conversion of a base First discovered in trypanosomes Now known to occur in many organisms Refer to Table 12.5 for a list of examples RNA editing 12-77

78. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA editing 12-78