Chapter 14: Gene Transcription and RNA Modification

Transcription

1 Chapter 14: Gene Transcription and RNA Modification Student Learning Objectives Upon completion of this chapter you should be able to: 1. Understand the role of transcription in gene expression. 2. Know the initiation, elongation, and termination stages of transcription in bacteria. 3. Understand how eukaryotic transcription differs from bacterial transcription. 4. Understand the importance of regulatory elements and transcription factors to eukaryotic transcription. 5. Understand the processes of RNA modification and why each of these processes is needed in a eukaryotic cell Overview of Transcription Overview The central dogma of molecular biology, also known as the central dogma of genetics (Figure 14.1), describes the process by which the genetic information is interpreted to form a functional protein. Gene expression is actually a two stage process. In this chapter we will examine the biochemical process of transcription. The basic purpose of transcription is to make a copy of the protein-building instructions of a gene. The next chapter will examine the process of translation, in which the instructions contained within the copy are used to construct a functional protein. This initial section of the chapter serves as an important overview to transcription. The terms and processes described within this section are used for both prokaryotic and eukaryotic transcription. You should master these terms before proceeding to the next sections. Outline of Key Terms Gene Structural genes Nonstructural genes Central dogma of genetics Gene expression Transcription mrna Translation Polypeptide Transcription stages Initiation Elongation Termination Gene organization Promoter Terminator Template strand Antisense strand Coding strand Sense strand Regulatory sequences Transcription factors mrna organization Ribosomal binding site Codons Start codon Stop codon 154

2 Focal Points Central dogma of genetics (Figure 14.1) Organization of a bacterial gene and its mrna transcript (Figure 14.2) Stages of transcription (Figure 14.3) Functions of RNA molecules (Table 14.1) Exercises and Problems For questions 1 to 7, match the term to its correct definition. 1. Terminator 2. Codons 3. Regulatory sequences 4. Promoter 5. Template stand 6. Gene 7. Structural genes a. Binding sites for proteins that control transcription. b. Contains genetic information that is complementary to the RNA transcript. c. Groups of three nucleotides within the mrna. d. The site that controls the start of transcription. e. The site that controls the end of transcription. f. A transcriptional unit that can be transcribed into RNA. g. Produces mrna when transcribed. For questions 8 to 11, indicate whether the statement is associated with the initiation (I), elongation (E), or termination (T) of transcription. 8. The RNA polymerase synthesizes a mrna transcript. 9. The RNA polymerase interacts with the promoter. 10. The DNA forms an open complex. 11. The RNA polymerase and mrna dissociate from the DNA. For each of the following statements, indicate the type of RNA from Table 14.1 that best matches the statement. 12. Targets the proteins to the endoplasmic reticulum of eukaryotes. 13. Small nuclear RNA that is involved in the splicing of eukaryotic mrna. 14. Contains the instructions for a protein sequence. 15. Cytoplasmic RNA of prokaryotes that is used in protein secretion. 16. A component of the ribosome. 17. Transfer RNA. 155

3 14.2 Transcription in Bacteria Overview This section outlines the process of transcription in bacteria. As was the case with replication, it is better to understand the simpler bacterial transcription system before proceeding to the more complex eukaryotic one. Before entering into the discussion of initiation, elongation, and termination, the section takes a good look at the structure of a bacterial promoter. A promoter is a regulatory sequence that plays a central role in transcriptional regulation. There are two things that you should focus on with regards to the promoter. The first is the numbering of nucleotides (Figure 14.4) and the second is the concept of a consensus sequence (Figure 14.5). Notice that not all promoters have the same nucleotide sequence, although they are very similar. The best way to understand transcription initiation, elongation, and termination is to first read the sections of the text, and then study the appropriate diagrams. Remember that these are three-dimensional systems, and it is a complex interaction of proteins and nucleic acids (both DNA and RNA) that makes the process possible. Outline of Key Terms Promoter Sequence elements Pribnow box Consensus sequence RNA polymerase Holoenzyme Core enzyme Sigma ( ) factor Helix-turn-helix motif Promoter complexes Closed complex Open complex Transcription termination -dependent termination Rho ( ) factor -independent termination Intrinsic termination Focal Points Transcription initiation (Figure 14.7) Synthesis of the RNA transcript (Figure 14.8) Transcription termination (Figures and 14.11) Exercises and Problems For questions 1 to 6, match the term with its correct definition. 1. Helix-turn-helix 2. Transcription factors 3. RNA polymerase 4. Holoenzyme 5. Closed complex 6. Promoter 156

4 a. An RNA polymerase that has sigma factor attached. b. The region of a gene between -10 and -35 that contains the Pribnow box. c. Occurs when the holoenzyme is bound to the promoter region. d. Proteins that influence the relationship of the holoenzyme with the promoter. e. A four subunit molecule that catalyzes the synthesis of RNA. f. A region of the sigma factor that facilitates binding to the DNA. For questions 7 to 14 indicate whether the statement is associated with initiation (I), elongation (E), or termination (T) of bacterial transcription. 7. Disrupts the hydrogen bonding between the DNA and RNA. 8. Starts with the formation of a closed complex. 9. May occur either dependently or independently of the rho protein. 10. Actively uses the template strand for RNA synthesis. 11. Unraveling of the DNA occurs in AT-rich regions 12. The RNA polymerase moves along the DNA. 13. RNA is synthesized in a 5 to 3 direction. 14. The sigma factor within the holoenzyme binds to the DNA Transcription in Eukaryotes Overview Transcription in eukaryotes is slightly more complicated than in bacteria, although the principles remain the same. During initiation, the RNA polymerase must still bind to the promoter and form an open complex. However, unlike the bacterial systems, eukaryotic systems employ three different RNA polymerases to transcribe the various categories of genes. These include RNA polymerase II, which transcribes mrnas. Moreover, eukaryotes utilize a variety of proteins, called transcription factors, which regulate the binding of the polymerase to the promoter (Figure 14.14). Other types of transcription factors bind regulatory elements and allow for a fine tuning of the transcription process. Also, note how the termination of transcription occurs after the mrna is released from the DNA template (Figure 14.15). Outline of Key Terms Core promoter TATA box Transcriptional start site Basal transcription Basal transcription apparatus General transcription factors Preinitiation complex Mediator Regulatory elements Enhancers Silencers Cis-acting elements DNA sequences Trans-acting factors Proteins 157

5 Focal Points Promoter regions of a eukaryotic structural gene (Figure 14.13) Interactions of transcription factors and RNA polymerase in initiation (Figure 14.14) Possible models for transcription termination (Figure 14.15) Exercises and Problems For questions 1 to 7, match each of the components of a eukaryotic structural gene to its correct definition. 1. trans-acting factor 2. TATA box 3. enhancer 4. core promoter 5. regulatory element 6. cis-acting element 7. silencer a. Any sequence that effects the ability of the RNA polymerase to bind the promoter. b. A regulatory sequence that inhibit the level of transcription. c. A general term for regulatory elements that influence the activity of a nearby gene. d. A regulatory sequence that increase the level of transcription. e. This will produce a low level of transcription even in the absence of regulatory elements. f. This determines the precise start site of transcription. g. A gene that controls a second gene from a distance. For each of the following components of the transcription preinitiation complex, match the protein with its correct function. 8. TFIIB 9. TFIID 10. TFIIF 11. Mediator 12. TFIIH 13. TFIIE a. Interacts with the transcription factors and the RNA polymerase. b. Serves as a junction between the RNA polymerase, TFIIB and the promoter. c. This protein binds to the TATA box in the promoter. d. Binds to TFIID, allows the RNA polymerase to bind to the core promoter. e. Plays the major role in the formation of the open complex. f. Maintains the open complex. 158

6 14.4 RNA Modification Overview In eukaryotic organisms, transcription does not directly produce a functional mrna. Instead, following transcription, the pre-mrna molecule is modified by a number of different processes that can drastically alter the size and function of the pre-mrna produced by the RNA polymerase (Table 14.3). These processes can be divided into two general categories. The first form of modification is a chemical change to the RNA transcript. This is usually in the form of 5 capping or 3 polya tailing, and sometimes even editing changes that alter the base sequence after it has been synthesized. The second form of modification involves alterations in the RNA length. This includes trimming of large transcripts into smaller pieces, and the removal of non-coding or intervening sequences. These sequences, called introns, are removed by RNA splicing. You need to familiarize yourself with all these modifications and the terminology associated with them. Outline of Key Terms Colinearity RNA splicing Exons Introns Intervening sequences rrna processing Nucleolus trna processing Endonuclease Exonuclease Ribozyme Self-splicing Group I introns Group II introns Maturases Spliceosome Pre-mRNA Heterogeneous nuclear RNA (hnrna) snrnps Alternative splicing Other RNA modifications Capping 5 cap Polyadenylation 3 poly A tail Editing Focal Points Overview of RNA modifications (Table 14.3) The processing of ribosomal RNA in eukaryotes (Figure 14.16) The processing of a precursor trna molecule in E. coli (Figure 14.17) RNA splicing (Figures ) Capping (Figure 14.21) PolyA tailing (Figure 14.22) RNA editing (Figure 14.23) 159

7 Exercises and Problems For questions 1 to 9, use the following answers. Some answers may be used more than once. a. 5 capping b. 3 polya tailing c. Splicing 1. May involve the use of snrnps. 2. Usually serves to shorten the length of the RNA transcript. 3. This process may occur while the transcript is being made by the RNA polymerase. 4. This plays an important role in the early stages of translation. 5. This provides stability to the mrna molecule. 6. Removes the introns from the pre-mrna. 7. May produce an mrna with different combinations of introns. 8. Identifies the consensus sequence AAUAAA in the pre-mrna. 9. Attaches a guanosine monophosphate to the pre-mrna. For questions 10 to 14, identify the type of splicing that is involved in the statement. a. group II b. spliceosome c. group I d. all of the above 10. Involves the use of snrnps. 11. Uses a free guanosine to enable splicing. 12. Produces a conformational change in the pre-mrna to enable splicing. 13. Identifies consensus sequences in the pre-mrna. 14. Utilizes an adenine nucleotide present within the intron. For questions 15 to 17, provide the correct term that completes the statement. 15. An enzyme that is composed of RNA, not protein, is called a. 16. In eukaryotes, the coding information for a protein is contained within the of a gene. These are separated by DNA sequences called that are not translated into protein. 17. In eukaryotes, one gene may produce many similar proteins due to the process of. Chapter Quiz 1. Which of the following would reduce the overall level of transcription? a. transcription factors b. enhancers c. silencers d. core promoter e. none of the above 160

8 2. Sigma factor is associated with transcription in. a. bacteria b. eukaryotes c. viruses d. all organisms 3. This process increases the stability of the mrna. a. 5 capping b. alternative splicing c. 3 polya tailing d. spliceosomes e. none of the above 4. The transcription factor that binds to the TATA sequence in eukaryotes is. a. sigma b. rho c. TFIIB d. TFIID e. mediator 5. This process involves the use of snrnps. a. 3 polya tailing b. initiation of transcription c. spliceosome activity d. transcription termination 6. The Pribnow box is located where? a. the bacterial promoter b. the core promoter of eukaryotes c. eukaryotic introns d. the end of the coding sequence e. none of the above 7. Which of the following is NOT associated with the initiation of transcription in eukaryotes? a. rho b. mediator c. formation of an open complex d. transcription factors e. RNA polymerase II 8. In RNA editing, the most common modification is a. decarboxylation. b. deamination. c. methylation. d. phosphorylation. 9. The allosteric and torpedo models have been proposed to describe which of the following? a. transcription initiation in bacteria b. transcription termination in bacteria c. transcription initiation in eukaryotes d. transcription termination in eukaryotes 161

9 10. The information for producing a functional protein is contained within the of a eukaryotic gene. a. introns b. exons c. polya tail d. 5 cap e. group I introns Answer Key for Study Guide Questions This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers in parentheses ( ) represent possible alternate answers to a problem, while answers marked with an asterisk (*) indicate that the response to the question may vary e 2. c 3. a 4. d 5. b 6. f f 2. d 3. e 4. a 5. c g 2. f 3. d 4. e 5. a c 2. c 3. a 4. a 5. b 6. c 7. g 8. E 9. I 10. I 11. T 12. 7S RNA 6. b 7. T 8. I 9. T 10. E 6. c 7. b 8. d 9. c 10. b 7. c 8. b 9. a 10. b 11. c 12. d 13. snrna 14. mrna 15. scrna 16. rrna 17. trna 11. I 12. E 13. E 14. I 11. a 12. e 13. f 13. d 14. a 15. ribozyme 16. exons; introns 17. alternative splicing Quiz 1. c 2. a 3. c 4. d 5. c 6. a 7. a 8. b 9. d 10. b 162