Principles of Biology
49
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Transcription Transcription is the process of copying information from DNA to RNA.
A scribe makes careful work. Similar to the way a scribe would make copies of one manuscript to another, transcription is the relay of information in DNA to a new but similar form, RNA. Jean Le Tavernier, portrait of Jean Miélot, after 1456.
Topics Covered in this Module Transcription versus DNA Replication
Major Objectives of this Module Explain the processes that occur during the three phases of transcription. Describe the molecular factors that aid in transcription. Relate the importance of specific sequences on the DNA molecule to the process of transcription. Describe the differences between eukaryotic and prokaryotic transcription. Describe RNA processing.
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Principles of Biology 49 Transcription
Transcription versus DNA Replication Each diploid cell contains only 2 copies of each gene, but needs to make a large amount of protein from the genetic information. The first step in this process is to create many copies of the genetic information as RNA instead of DNA. The process of transcription creates an RNA version of the information coded in the DNA. Transcription is similar to DNA replication in that the DNA is unwound and a polymerase reaction adds the appropriate nucleotide substrates to the growing nucleotide chain. However, there are several key differences between DNA replication and transcription. During transcription, only one strand of the DNA is used as a template to create the RNA molecule. This is called the template strand. The other strand is called the non-template or coding strand. It is called the coding strand because its sequence will match the sequence of the newly created RNA strand, except that the RNA will contain the nucleotide uracil (U) in place of thymine (T) in the DNA. The enzyme that performs the polymerase reaction in transcription is called RNA polymerase. Bacteria have one type of RNA polymerase while eukaryotes have at least three. RNA polymerase I transcribes genes that code for the large RNA molecules, called ribosomal RNA (rRNA), that are found in ribosomes. RNA polymerase II transcribes protein-coding genes and creates messenger RNA (mRNA). RNA polymerase III transcribes genes that code for transfer RNAs (tRNAs) that play a key role during translation. In addition to these, new RNA polymerases that produce RNA involved in regulation of gene expression have recently been identified. RNA polymerase moves 3′ to 5′ along the template strand of the DNA and synthesizes the RNA molecule in the 5′ to 3′ direction. Using the coding strand as a reference, sequences that are on the 5′-side of a reference point are called "upstream," and sequences on the 3′-side are called "downstream." Unlike DNA polymerase, RNA polymerase does not need a primer to start transcription. The stretch of DNA that is transcribed into RNA is known as the transcription unit. Transcription has three distinct phases: initiation, elongation and termination. During initiation, with the help of additional factors, RNA polymerase binds to the DNA and unwinds it. During the elongation phase, RNA polymerase moves along the DNA template and creates the RNA transcript. Finally, termination occurs when RNA polymerase reaches the termination site and the RNA transcript is released. The initiation of transcription requires a special DNA sequence called a promoter. The promoter tells the RNA polymerase where to start transcription and is positioned upstream of the transcription start site, also known as the +1 site because it is the site at which the first RNA nucleotide is added. The promoter also tells RNA polymerase which DNA strand to use as the template. The sequences and factors involved in initiation differ between prokaryotic and eukaryotic transcription. Transcription differs in prokaryotes and eukaryotes. In prokaryotes, promoters are between 40–50 base pairs long and they include a six-base-pair sequence identical or similar to TATAAT. This sequence is located approximately 10 base pairs upstream from the +1 site and is known as the -10 box. A second key sequence, TTGACA, occurs 35
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base pairs upstream from the +1 site, and is therefore called the -35 box. While most prokaryotic promoters include both a -10 box and a -35 box, the promoter sequences outside of these regions vary widely. The sequences in eukaryotic promoters are more diverse than prokaryotic promoters. Despite the increase in diversity, many eukaryotic promoters for protein-coding genes have a similar structure for their "core" promoter. One element of the core promoter — called the TATA box — is located 25–30 base pairs upstream from the transcription start site. Another consensus site, the TFIIB recognition element, is often located in the promoter region at approximately 35 base pairs upstream from the transcription start site. Finally, the core promoter may also include an initiator element centered on the transcription start site and a downstream core promoter element roughly 30 base pairs downstream of the +1 site (Figure 1).
Figure 1: Eukaryotic Promoter Structure. Several consensus sequences are found in the core promoter region of a gene that codes for a protein. Not all of these sequences are found in every promoter. A transcription start site consists of a core promoter element and a regulatory promoter. The core promoter elements include the TFIIB recognition element, the TATA box, the initiator element and the downstream core promoter element. © 2011 Nature Education All rights reserved. Eukaryotes also use enhancer sequences, which increase the efficiency of transcription initiation of the corresponding gene. Enhancers may be located hundreds or thousands of base pairs from the promoter and are brought to the promoter by DNA looping. This looping is facilitated by proteins known as activators. Proteins that inhibit looping are called repressors. In addition to RNA polymerase, there are other factors that are required for transcription. In prokaryotes, a protein subunit called sigma binds to the core RNA polymerase to create what is known as the RNA polymerase holoenzyme. It is the sigma portion of the holoenzyme that binds to the promoter to initiate transcription. There are a variety of sigma proteins, each with a slightly different structure. By pairing with different sigma proteins, RNA polymerase may bind to different promoters. The genes transcribed by the holoenzyme are dependent on which sigma protein is present in the holoenzyme. Eukaryotes also require additional factors for RNA polymerase to bind to the DNA. These proteins are called the general transcription factors. These proteins assemble at the promoter first, and then RNA polymerase binds to form what is known as the transcription initiation complex. Once the holoenzyme (in prokaryotes) or transcription initiation complex (in eukaryotes) is bound to the promoter, the DNA helix unwinds, exposing approximately 13 base pairs at a time. Using the template strand of DNA, RNA polymerase begins adding nucleotide monomers to the growing transcript. Once approximately 10 nucleotides are polymerized, initiation is considered complete and elongation begins.
Test Yourself If a mutation changed the sequence of the -10 box, what would you expect the result to be?
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BIOSKILL DNA-RNA Hybridization How do scientists determine which DNA sequences are bound by transcription factor proteins? Protein-DNA interactions are important for transcription, DNA replication, and many other biological processes, and it is important to understand where along the DNA the protein is binding. One of the laboratory techniques that scientists use to study protein-DNA interactions is chromatin immunoprecipitation (ChIP) (Figure 2). class="NoSpacing" >The first step in ChIP is to cross-link the protein-DNA complexes in the cell using a cross-linking agent, such as formaldehyde. This will maintain the association of the protein with the DNA so that the entire complex can be isolated. The DNA is then physically disrupted or enzymatically digested into approximately 500-base-pair pieces. The pieces of protein-bound DNA are then isolated using an antibody highly specific for the protein of interest and precipitated away from protein-DNA complexes not containing the protein of interest. class="NoSpacing" >Cross-linking of the immunoprecipitated protein-DNA sample is reversed by breaking the bonds between the protein and DNA. The DNA that was isolated with the protein is purified and analyzed using one of several techniques, including quantitative PCR, sequencing, or microarray. This allows scientists to identify which DNA sequences are directly bound to the protein of interest. class="NoSpacing" >
Figure 2: Steps of the chromatin immunoprecipitation (ChIP) procedure. In a ChIP procedure, bound protein is used to isolate the DNA sequences recognized by the protein. In this example, Caenorhabditis elegans genomic DNA sequences are bound to specific regulatory proteins, and these complexes are cross-linked, immunoprecipitated, and purified. The DNA sequences can be analyzed by PCR, microarray, cloning or Southern blotting. © 2013 Nature Education All rights reserved.
BIOSKILL Elongation, termination, and processing create the final RNA transcript. During elongation, RNA polymerase moves along the DNA template 3′ to 5′ and adds new nucleotides to the 3′ end of the RNA transcript (Figure 3). Nucleotides are added to the RNA by complementary base pairing to the DNA template strand. The base pairing during transcription is the same as in DNA base pairing, except that RNA contains uracil instead of thymine. Therefore, RNA polymerase uses the nucleotides CTP, GTP, ATP, and UTP to create the transcript. RNA polymerase catalyzes the formation of phosphodiester bonds between these monomers as the transcript is created, at a rate of approximately 40 nucleotides per second. As transcription continues along the DNA, the RNA transcript separates from the DNA template and the DNA double helix is re-formed (Figure 4). A single gene may produce many RNA transcripts at the same time. Once one RNA polymerase molecule begins the elongation phase, initiation may occur with another RNA polymerase molecule. By having many copies of RNA created at the same time, the cell is capable of generating a large amount of RNA from a single gene very quickly.
Figure 3: Elongation. Many copies of RNA can be transcribed at one time. Here, an electron micrograph shows RNA branching like leaf veins off the central spoke of DNA. The strand to the left has numerous filamentous protrusions, which represent the transcription of numerous copies of RNA. The other two DNA strands lack the filamentous protrusions and are not being transcribed. © 2002 Nature Publishing Group
Dragon, F., et al. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417, 967-970 (2002) doi:10.1038/nature00769. Used with permission. Figure 4: The transcription process. Test your understanding of how transcription works.
© 2014 Nature Education All rights reserved.
Transcript
Termination occurs once RNA polymerase reaches a specific sequence on the DNA template. In bacteria, one type of terminator sequence codes for a stretch of RNA that, once transcribed, creates a hairpin loop by folding back on itself. The short hairpin is created by base pairing of complementary G-C bases within the RNA. The region downstream of the hairpin is rich in A bases in the DNA — and therefore U bases in the RNA. The formation of the stronger G-C base pairs in the hairpin, combined with the weaker U-A base pairing between RNA and DNA in the downstream region, disrupts the association between the RNA polymerase, the DNA template, and the RNA transcript. In eukaryotes, termination occurs when a sequence called a polyadenylation signal (AAUAAA) is transcribed. Once RNA polymerase reaches 10-35 base pairs downstream of the polyadenylation signal, the RNA transcript is released from RNA polymerase. Test Yourself Describe the three phases of transcription.
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Test Yourself Which phases of transcription could be affected by changes to DNA sequences?
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In bacteria, the RNA is ready for translation as soon as it is transcribed. However, in eukaryotes the mRNA must undergo processing in the nucleus before translation may begin in the cytoplasm. Before this processing occurs, the mRNA transcript is known as pre-mRNA. The ends of the pre-mRNA are modified in specific ways. After the first 20–40 nucleotides of the pre-mRNA are synthesized during transcription elongation, a modified guanine (G) nucleotide is added to the 5′ end of the transcript, creating the 5′ cap. The 5′ cap helps the transcript bind to the ribosome for translation. It also helps protect the mRNA from enzymatic degradation in the cytoplasm. A poly(A) tail is added to the 3′ end of the pre-mRNA transcript. The poly(A) tail is made up of 50–300 adenine (A) nucleotides. The poly(A) tail aids in the export of the mRNA to the cytoplasm for translation, and, like the 5′ cap, the poly(A) tail protects the mRNA from degradation. The current estimate is that there are approximately 20,000 human genes. However, human cells make over 75,000 proteins. How is that possible? The answer lies in a process known as RNA splicing. In eukaryotes, large portions of the pre-mRNA molecule are removed before the mRNA is exported from the nucleus. The segments of the pre-mRNA that are included in the final mRNA molecule are called exons. The non-coding segments that are removed are called introns (Figure 5). There is a consensus sequence at the junctions between exons and introns. These short sequences of DNA have little variation between different genes.
Figure 5: RNA Splicing. In eukaryotes, before translation can occur, introns must be removed and the exons combined to form mature mRNA. © 2012 Nature Education All rights reserved. Once the pre-mRNA is transcribed, several small nuclear ribonucleoprotein particles (snRNPs) bind to the consensus sequences. Other proteins also associate to form an RNA-protein complex known as a spliceosome. A
spliceosome is created at each exon-intron junction. The spliceosome cuts the pre-mRNA, removes the intron and joins the exons together (Figure 6). In this way, all of the introns are removed, and exons are spliced together to form the mature mRNA that is ready for translation.
Figure 6: snRNPs combine to form the spliceosome, which facilitates RNA splicing. Small nuclear ribonucleoproteins (snRNPs) are complexes of RNA and protein that bind to a pre-mRNA to be spliced. The pre-mRNA contains three sites critical to the splicing process: the 5′ splice site, the 3′ splice site, and the branch point, which usually includes an adenine base. In the first step, snRNPs bind to the 5′ splice site and branch point. The snRNPs are brought together, allowing the branch point to cut the 5′ splice site from the adjacent exon. Next, three more snRNPs bind to the pre-mRNA, forming the complete spliceosome. The spliceosome folds the intron into a looped structure called a lariat. In the last step, the spliceosome brings the exons together and covalently links them. At the same time, the lariat of intronic RNA is released. © 2014 Nature Education All rights reserved. Most introns do not have a known specific function, though some contain regulatory sequences that affect gene expression. One effect of RNA splicing is the ability to change which sequences are treated as exons and therefore create different mature mRNA molecules from the same gene. This is known as alternative RNA splicing.
Proteins are made up of structural and functional regions called domains. For example, one domain of an enzyme may contain an active site while another may contain an allosteric site. In many cases, different domains are coded for by different exons. By using alternative splicing, the same gene is able to produce a variety of proteins that contain different domains. Test Yourself Describe three ways that the RNA transcript is modified prior to translation in eukaryotes.
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IN THIS MODULE
Transcription versus DNA Replication Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete? Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Master gene KLF14 controls genes in metabolic syndrome Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. View | Download Interfering with microRNAs to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. View | Download
SCIENCE ON THE WEB
Nature Milestone: DNA Technology A collection of articles from Nature Publishing Group about DNA and how scientists study its form and function How Did We Discover Transcription? A collection of research papers covering seminal discoveries about transcription
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Principles of Biology 49 Transcription
Summary Explain the processes that occur during the three phases of transcription. During initiation, RNA polymerase and additional factors bind to the DNA and unwind it to access the template strand of DNA. During elongation, RNA polymerase adds nucleotide monomers to the growing RNA strand. Termination involves the disruption of the association between RNA polymerase, the DNA template and the RNA transcript. OBJECTIVE
Describe the molecular factors that aid in transcription. RNA polymerase catalyzes the polymerization reaction that adds nucleotide monomers to the growing RNA molecule. Additional factors are required for the initiation of transcription, including the sigma protein in bacteria and a variety of transcription factors, in eukaryotes. OBJECTIVE
Relate the importance of specific sequences on the DNA molecule to the process of transcription. Specific sequences in the DNA, such as the core promoter elements and enhancers, help bring RNA polymerase to the transcription start site. Termination is also signaled by specific sequences in the DNA that result in the formation of a hairpin loop that disrupts the association between the DNA template and the RNA transcript. OBJECTIVE
Describe the differences between eukaryotic and prokaryotic transcription. Prokaryotes and eukaryotes use different promoter sequences and additional factors to initiate transcription. Different termination sequences and mechanisms are also used. In prokaryotes, the RNA transcript is ready for translation as soon as it is created. In eukaryotes, RNA processing must occur before the RNA transcript is exported from the nucleus and is ready for translation. OBJECTIVE
Describe RNA processing. In eukaryotes, a 5′ cap and a 3′ poly(A) tail are added to the pre-mRNA. The introns are spliced out to create a mature mRNA molecule that contains only exons. The mature mRNA is now ready to be exported from the nucleus. OBJECTIVE
Key Terms elongation The phase of transcription in which RNA polymerase moves along the DNA template and incorporates nucleotides into the growing RNA transcript. initiation The phase of transcription in which RNA polymerase binds to the promoter of a gene and begins RNA synthesis. intron A segment of mRNA that is removed prior to translation. promoter The DNA sequence at which RNA polymerase binds to initiate transcription of a gene. RNA polymerase An enzyme that uses a DNA template to synthesize a complementary RNA molecule during transcription.
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TATA box An element of the core promoter in eukaryotic genes; contains the consensus sequence TATAAA. termination The phase of transcription in which RNA polymerase releases the RNA transcript and detaches from the DNA template. transcription factor A protein that regulates the transcription of specific genes. transcription initiation complex The combination of transcription factors and RNA polymerase that assembles at the promoter of a gene prior to transcription initiation. transcription start site The site at which the first RNA nucleotide is added; also known as the +1 site. IN THIS MODULE
Transcription versus DNA Replication Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete? Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Master gene KLF14 controls genes in metabolic syndrome Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. View | Download Interfering with microRNAs to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. View | Download
SCIENCE ON THE WEB
Nature Milestone: DNA Technology A collection of articles from Nature Publishing Group about DNA and how scientists study its form and function How Did We Discover Transcription? A collection of research papers covering seminal discoveries about transcription
page 254 of 989
1 pages left in this module
Principles of Biology 49 Transcription
Test Your Knowledge 1. Which specific sequence is important for termination in eukaryotes?
5′ cap sigma TFIIB a polyadenylation signal TATA box
2. Which of the following does NOT occur during transcription in bacteria?
The holoenzyme binds the promoter. Termination sequence signals cause the RNA to be released. RNA splicing Uracil is used instead of thymine. RNA polymerase adds monomers to the growing RNA transcript.
3. Which of the following is important for transcription in prokaryotes?
RNA splicing TATA box sigma RNA polymerase III spliceosomes
4. If the template strand of DNA has the sequence 3′-TCTAGGACT-5′, what will the
sequence of the transcribed RNA be? 5′-AGATCCTGA-3′ 5′-AGAUCCUGA-3′ 5′-UCAGGAUCU-3′ 5′-TCAGGATCT-3′ 5′-UCAGGATCT-3′
5. Other than the core RNA polymerase, what proteins are required for the initiation of
transcription in bacteria? enhancers sigma promoters a variety of transcription factors activators
6. Mutation of which of these sequences would have no effect on the initiation of
transcription? Polyadenylation signal TFIIB recognition element initiator element downstream core promoter element TATA box
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7. If the coding strand of DNA has the sequence 5′-CGAGACTTCTGA-3′, what will
the sequence of the transcribed RNA be? 5′-CGUGUCTTCTGU-3′ 5′-CGAGACUUCUGA-3′ 3′-GCTCTGAAGACT-5′ 3′-GCUCUGAAGACU-5′ 5′-CGAGACTTCTGA-3′
Submit
IN THIS MODULE
Transcription versus DNA Replication Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential? Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete? Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Master gene KLF14 controls genes in metabolic syndrome Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. View | Download Interfering with microRNAs to control gene expression Silencing of microRNA families by seed-targeting tiny LNAs. View | Download Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. View | Download
SCIENCE ON THE WEB
Nature Milestone: DNA Technology A collection of articles from Nature Publishing Group about DNA and how scientists study its form and function How Did We Discover Transcription? A collection of research papers covering seminal discoveries about transcription
page 255 of 989
