PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 12 GENE TRANSCRIPTION AND RNA MODIFICATION Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

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

The central dogma of genetics Figure 12.1 12-5

12.1 OVERVIEW OF TRANSCRIPTION Gene expression is the overall process by which the information within a gene is used to produce a functional product which can determine a trait in play with the environment Figure 12.2 shows common organization of a gene 12-6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Signals the end of protein synthesis common organization of a gene Signals the end of protein synthesis 12-7

Gene Expression Requires Base Sequences The strand that is actually transcribed (used as the template) is termed the template strand The opposite strand is called the coding strand or the sense 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 12-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Figure 12.3 12-10

RNA Transcripts Have Different Functions 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 12-12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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12.2 TRANSCRIPTION IN BACTERIA 12-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

12-16 Figure 12.4 The conventional numbering system of promoters Most of the promoter region is labeled with negative numbers 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 Figure 12.4 The conventional numbering system of promoters 12-16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Sequence elements that play a key role in transcription The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level Sequence elements that play a key role in transcription Sometimes termed the Pribnow box, after its discoverer Figure 12.4 The conventional numbering system of promoters 12-17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 The most commonly occurring bases Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters 12-18 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Initiation of Bacterial Transcription 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 = a2bb’ Sigma factor One subunit = s These subunits play distinct functional roles 12-19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Initiation of Bacterial Transcription 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 12-20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements Figure 12.6 12-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

A short RNA strand is made within the open complex 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 12-22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.7 12-23

Elongation in Bacterial Transcription 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 12.8 depicts the key points in the synthesis of the RNA transcript 12-25 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Similar to the synthesis of DNA via DNA polymerase Figure 12.8 12-26

Termination of Bacterial Transcription 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 r (rho) 2. rho-independent termination Does not require r 12-27 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Rho protein is a helicase rho utilization site Rho protein is a helicase r-dependent termination Figure 12.10 12-28

r-dependent termination Figure 12.10 12-29

r-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 Us Stabilizes the RNA pol pausing URNA-ADNA hydrogen bonds are very weak No protein is required to physically remove the RNA from the DNA This type of termination is also called intrinsic r-independent termination Figure 12.11 12-30

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 organisms and cells Cellular complexity such as organelles added complexity means more genes Multicellularity increased regulation to express only in right cells at right time 12-31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic RNA Polymerases 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 12-32 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic RNA Polymerases 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 12.12 12-33 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Sequences of Eukaryotic Structural Genes 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 12-34 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The core promoter is relatively short It consists of the TATA box 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 12-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.13 Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types Enhancers Stimulate transcription Silencers Inhibit transcription They vary widely in their locations but are often found in the –50 to –100 region 12-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Sequences of Eukaryotic Structural Genes 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 12-37 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

RNA Polymerase II and its Transcription Factors 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 12-38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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12-40 Figure 12.14 A closed complex CTD carboxy terminal domain (CTD) RNA pol II can now proceed to the elongation stage Released after the open complex is formed 12-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Basal transcription apparatus RNA pol II + the five GTFs The third component 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 appears to regulate the ability of TFIIH to phosphorylate CTD Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation 12-41 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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Chromatin Structure and Transcription 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 12-43 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromatin Structure and Transcription 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 12-44 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Removes acetyl groups, thereby restoring a tighter interaction 1. Covalent modification of histones Amino terminals of histones are modified in various ways Acetylation; phosphorylation; methylation Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction Figure 12.15 12-45 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 Proteins are members of the SWI/SNF family Acronyms refer to the effects on yeast when these enzyme are defective Mutants in SWI are defective in mating type switching Mutants in SNF are sucrose non-fermenters These effects may significantly alter gene expression Figure 12.15 12-46 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 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 12-47 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 12-48 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 12-49 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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Processing 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 12-51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-52 Figure 12.16 This processing occurs in the nucleolus Functional RNAs that are key in ribosome structure Figure 12.16 12-52 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Processing 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 This event has been studied extensively in E. coli Figure 12.17 shows the trimming of a precursor tRNA that carries the amino acid tyrosine (tRNAtyr) Interestingly, the cleavage occurs differently at the 5’ end and the 3’ end 12-53 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-54 Figure 12.17 Found to contain both RNA and protein subunits Covalently modified bases However, RNA contains the catalytic ability Therefore, it is a ribozyme Figure 12.17 12-54 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 b-globin gene It had been cloned earlier Leder used a strategy that involved hybridization 12-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Experiment 12A: Identification of Introns Via Microscopy Double-stranded DNA of the cloned b-globin gene is first denatured Then mixed with mature b-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 12-56 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-57 Figure 12.18 RNA displacement loop mRNA cannot hybridize to this region Because the intron has been spliced out from the mRNA Figure 12.18 12-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the Hypothesis The b-globin gene from the mouse contains one or more introns Testing the Hypothesis Refer to Figure 12.19 12-58 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.19 12-59

The Data 12-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

This suggests that the b-globin gene contains introns Interpreting the Data Hybridization caused the formation of two R loops, separated by a double-stranded DNA region This suggests that the b-globin gene contains introns 12-61 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Splicing 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 12-62 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 12.20 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 12-63 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.20 12-64 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 Figure 12.20 12-65 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-66 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Pre-mRNA Splicing 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 12-67 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Pre-mRNA Splicing 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 12-68 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The pre-mRNA splicing mechanism is shown in Figure 12.22 Intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21 Corresponds to the boxed adenine in Figure 12.22 Sequences shown in bold are highly conserved Figure 12.21 Serve as recognition sites for the binding of the spliceosome The pre-mRNA splicing mechanism is shown in Figure 12.22 12-69 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Intron loops out and exons brought closer together Figure 12.22 12-70 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Intron will be degraded and the snRNPs used again Figure 12.22 12-71 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Intron Advantage? 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 12-72 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Intron Advantage? 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 12-73 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Capping 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 12.23, capping is a three-step process 12-74 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.23 12-75 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 12.23 12-76 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Capping 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 12-77 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

12-79 Figure 12.24 Consensus sequence in higher eukaryotes Length varies between species Appears to be important in the stability of mRNA and the translation of the polypeptide From a few dozen adenines to several hundred 12-79 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display