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CHAPTER 12 GENE TRANSCRIPTION AND RNA MODIFICATION.

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1 CHAPTER 12 GENE TRANSCRIPTION AND RNA MODIFICATION

2 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 OVERVIEW OF TRANSCRIPTION 12-3

3 Figure 12.1 12-5 Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides Start codon: specifies the first amino acid in a protein sequence, usually a formylmethionine (in bacteria) or a methionine (in eukaryotes) Signals the end of protein synthesis

4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The strand that is actually transcribed is termed the template strand (top in my diagrams) The opposite strand is called the coding strand or the sense strand (bottom in my diagrams) The base sequence is identical to the RNA transcript Except for the substitution of uracil in RNA for thymine in DNA Gene Expression Requires Base Sequences 12-6

5 12-8 The promoter functions as a recognition site for transcription factors The transcription factors enable RNA polymerase to bind to the promoter forming a closed promoter complex Following binding, the DNA is denatured into a bubble known as the open promoter complex, or simply an open complex Initiation Elongation RNA polymerase slides along the DNA in an open complex to synthesize the RNA transcript Termination A termination signal is reached that causes RNA polymerase to dissociated from the DNA Figure 12.2 The 3 Stages of Transcription

6 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 A structural gene is a one that encodes a polypeptide When such genes are transcribed, the product is an RNA transcript called messenger RNA (mRNA) Well over 90% of all genes are structural genes RNA Transcripts Have Different Functions 12-9

7 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 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 12-10

8 12-11

9 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 12.3 Promoters 12-13 TRANSCRIPTION IN BACTERIA

10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-14 Figure 12.3 The conventional numbering system of promoters Bases preceding this are numbered in a negative direction There is no base numbered 0 Bases to the right are numbered in a positive direction Sometimes termed the Pribnow box, after its discoverer Sequence elements that play a key role in transcription

11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-15 Figure 12.4 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

12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-18 Figure 12.5 Amino acids within the  helices hydrogen bond with bases in the promoter sequence elements Initiation of transcription - binding of RNA polymerase

13 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 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 12-19

14 12-20 Figure 12.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15 The RNA transcript is synthesized during the elongation step The DNA strand used as a template for RNA synthesis is termed the template or noncoding 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 12-21

16 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 the double helix On average, the rate of RNA synthesis is about 43 nucleotides per second! Figure 12.7 depicts the key points in the synthesis of the RNA transcript Elongation in Bacterial Transcription 12-22

17 12-23 Similar to the synthesis of DNA via DNA polymerase Figure 12.7

18 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 Termination of Bacterial Transcription 12-24

19 Many of the basic features of gene transcription are very similar in bacteria and eukaryotes However, gene transcription in eukaryotes is more complex Larger organisms Cellular complexity Multicellularity Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.3 TRANSCRIPTION IN EUKARYOTES 12-28

20 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 12-29

21 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 12.10 Eukaryotic RNA Polymerases 12-30

22 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 Transcriptional start site TATA box Regulatory elements Refer to Figure 12.11 Sequences of Eukaryotic Structural Genes 12-31

23 12-32 Usually an adenine The core promoter is relatively short It consists of the TATA box 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 12.11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

24 12-33 Figure 12.11 Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types Enhancers Stimulate transcription Silencers Inhibit transcription They vary 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 Usually an adenine

25 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 on nearby genes Example: TATA box, enhancers and silencers trans-acting elements Regulatory proteins that bind to such DNA sequences Sequences of Eukaryotic Structural Genes 12-34

26 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 12.12 shows the assembly of transcription factors and RNA polymerase II at the TATA box RNA Polymerase II and its Transcription Factors 12-35

27 12-36 Figure 12.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

28 12-37 Figure 12.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A closed complex TFIIH plays a major role in the formation of the open complex It has several subunits that perform different functions One subunit hydrolyzes ATP and phosphorylates a domain in RNA pol II known as the carboxyl terminal domain (CTD) This releases the contact between TFIIB and RNA pol II Other subunits act as helicases Promote the formation of the open complex Released after the open complex is formed RNA pol II can now proceed to the elongation stage

29 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The compaction of DNA to form chromatin can be an obstacle to the transcription process Most transcription occurs in interphase Then, chromatin is found in 30 nm fibers that are organized into radial loop domains Within the 30 nm fibers, the DNA is wound around histone octamers to form nucleosomes Chromatin Structure and Transcription 12-40

30 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol To circumvent this problem, the chromatin structure is significantly loosened during transcription Two common mechanisms alter chromatin structure Chromatin Structure and Transcription 12-41

31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 1. Covalent modification of histones Amino terminals of histones are modified in various ways Acetylation; phosphorylation; methylation 12-42 Figure 12.13 Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction

32 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible 12-43 Figure 12.13 These effects may significantly alter gene expression

33 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 This in turn corresponds to the sequence of amino acid 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 12.4 RNA MODIFICATION 12-44

34 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 12.4 RNA MODIFICATION 12-45

35 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 See Next Figure…. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.4 RNA MODIFICATION 12-46

36 12-47

37 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 12.14 shows the processing of mammalian ribosomal RNA Trimming 12-48

38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-49 Figure 12.14 Functional RNAs that are key in ribosome structure This processing occurs in the nucleolus

39 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

40 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 reconnected Refer to Figure 12.18 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

41 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-61 Figure 12.18

42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-62 Figure 12.16 In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA Also as heterogeneous nuclear RNA (hnRNA) 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

43 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most mature mRNAs have a 7-methyl guanosine 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 12.19, capping is a three-step process Capping 12-64

44 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 12-67

45 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 12.20 Tailing 12-68

46 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-69 Figure 12.20 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

47 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 12-70

48 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 12-71

49 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-72 Figure 12.21 Intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries The consensus sequences 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

50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-73 Intron loops out and exons brought closer together Figure 12.22

51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-74 Figure 12.22 Intron will be degraded and the snRNPs used again

52 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? 12-75

53 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? 12-76


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