Principles of Biology contents 49 Transcription17) 49-Transcription.pdf · Principles of Biology contents page 252 of 989 3 pages left in this module 49 Transcription Transcription

contentsPrinciples of Biology

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49 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 informationin 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.


49 Transcription

Transcription versus DNA ReplicationEach diploid cell contains only 2 copies of each gene, but needs to make alarge amount of protein from the genetic information. The first step in thisprocess is to create many copies of the genetic information as RNA insteadof DNA.

The process of transcription creates an RNA version of the informationcoded in the DNA. Transcription is similar to DNA replication in that the DNAis unwound and a polymerase reaction adds the appropriate nucleotidesubstrates to the growing nucleotide chain. However, there are several keydifferences between DNA replication and transcription.

During transcription, only one strand of the DNA is used as a template tocreate the RNA molecule. This is called the template strand. The otherstrand is called the non-template or coding strand. It is called the codingstrand because its sequence will match the sequence of the newly createdRNA strand, except that the RNA will contain the nucleotide uracil (U) inplace of thymine (T) in the DNA.

The enzyme that performs the polymerase reaction in transcription is calledRNA polymerase. Bacteria have one type of RNA polymerase whileeukaryotes have at least three. RNA polymerase I transcribes genes thatcode for the large RNA molecules, called ribosomal RNA (rRNA), that arefound in ribosomes. RNA polymerase II transcribes protein-coding genesand creates messenger RNA (mRNA). RNA polymerase III transcribes genesthat code for transfer RNAs (tRNAs) that play a key role during translation. Inaddition to these, new RNA polymerases that produce RNA involved inregulation of gene expression have recently been identified.

RNA polymerase moves 3′ to 5′ along the template strand of the DNA andsynthesizes the RNA molecule in the 5′ to 3′ direction. Using the codingstrand as a reference, sequences that are on the 5′-side of a reference pointare called "upstream," and sequences on the 3′-side are called"downstream." Unlike DNA polymerase, RNA polymerase does not need aprimer to start transcription. The stretch of DNA that is transcribed into RNAis known as the transcription unit.

Transcription has three distinct phases: initiation, elongation and termination.During initiation, with the help of additional factors, RNA polymerase bindsto the DNA and unwinds it. During the elongation phase, RNA polymerasemoves along the DNA template and creates the RNA transcript. Finally,termination occurs when RNA polymerase reaches the termination site andthe RNA transcript is released.

The initiation of transcription requires a special DNA sequence called apromoter. The promoter tells the RNA polymerase where to starttranscription and is positioned upstream of the transcription start site, alsoknown as the +1 site because it is the site at which the first RNA nucleotideis added. The promoter also tells RNA polymerase which DNA strand to useas the template. The sequences and factors involved in initiation differbetween prokaryotic and eukaryotic transcription.

Transcription differs in prokaryotes and eukaryotes.In prokaryotes, promoters are between 40–50 base pairs long and theyinclude a six-base-pair sequence identical or similar to TATAAT. Thissequence is located approximately 10 base pairs upstream from the +1 siteand is known as the -10 box. A second key sequence, TTGACA, occurs 35

Figure 1: Eukaryotic Promoter Structure.

© 2011 Nature Education All rights reserved.

Several consensus sequences are found in the core promoter region of agene that codes for a protein. Not all of these sequences are found inevery promoter. A transcription start site consists of a core promoterelement and a regulatory promoter. The core promoter elements includethe TFIIB recognition element, the TATA box, the initiator element and thedownstream core promoter element.

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, thepromoter sequences outside of these regions vary widely.

The sequences in eukaryotic promoters are more diverse than prokaryoticpromoters. Despite the increase in diversity, many eukaryotic promoters forprotein-coding genes have a similar structure for their "core" promoter. Oneelement of the core promoter — called the TATA box — is located 25–30base pairs upstream from the transcription start site. Another consensus site,the TFIIB recognition element, is often located in the promoter region atapproximately 35 base pairs upstream from the transcription start site.Finally, the core promoter may also include an initiator element centered onthe transcription start site and a downstream core promoter element roughly30 base pairs downstream of the +1 site (Figure 1).

Eukaryotes also use enhancer sequences, which increase the efficiency oftranscription initiation of the corresponding gene. Enhancers may be locatedhundreds or thousands of base pairs from the promoter and are brought tothe promoter by DNA looping. This looping is facilitated by proteins known asactivators. Proteins that inhibit looping are called repressors.

In addition to RNA polymerase, there are other factors that are required fortranscription. In prokaryotes, a protein subunit called sigma binds to the coreRNA polymerase to create what is known as the RNA polymeraseholoenzyme. It is the sigma portion of the holoenzyme that binds to thepromoter to initiate transcription. There are a variety of sigma proteins, eachwith a slightly different structure. By pairing with different sigma proteins,RNA polymerase may bind to different promoters. The genes transcribed bythe holoenzyme are dependent on which sigma protein is present in theholoenzyme.

Eukaryotes also require additional factors for RNA polymerase to bind to theDNA. These proteins are called the general transcription factors. Theseproteins assemble at the promoter first, and then RNA polymerase binds toform what is known as the transcription initiation complex.

Once the holoenzyme (in prokaryotes) or transcription initiation complex (ineukaryotes) is bound to the promoter, the DNA helix unwinds, exposingapproximately 13 base pairs at a time. Using the template strand of DNA,RNA polymerase begins adding nucleotide monomers to the growingtranscript. Once approximately 10 nucleotides are polymerized, initiation isconsidered complete and elongation begins.

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BIOSKILL

Test Yourself

If a mutation changed the sequence of the -10 box, what would you expect the result to be?

DNA-RNA HybridizationHow do scientists determine which DNA sequences are bound bytranscription factor proteins? Protein-DNA interactions are important fortranscription, DNA replication, and many other biological processes, and it isimportant to understand where along the DNA the protein is binding. One ofthe laboratory techniques that scientists use to study protein-DNAinteractions is chromatin immunoprecipitation (ChIP) (Figure 2).

class="NoSpacing" >The first step in ChIP is to cross-link the protein-DNAcomplexes in the cell using a cross-linking agent, such as formaldehyde.This will maintain the association of the protein with the DNA so that theentire complex can be isolated. The DNA is then physically disrupted orenzymatically digested into approximately 500-base-pair pieces. The piecesof protein-bound DNA are then isolated using an antibody highly specific forthe protein of interest and precipitated away from protein-DNA complexesnot containing the protein of interest.

class="NoSpacing" >Cross-linking of the immunoprecipitated protein-DNAsample is reversed by breaking the bonds between the protein and DNA.The DNA that was isolated with the protein is purified and analyzed usingone of several techniques, including quantitative PCR, sequencing, ormicroarray. This allows scientists to identify which DNA sequences aredirectly 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 sequencesrecognized by the protein. In this example, Caenorhabditis elegans genomicDNA sequences are bound to specific regulatory proteins, and thesecomplexes are cross-linked, immunoprecipitated, and purified. The DNAsequences can be analyzed by PCR, microarray, cloning or Southernblotting.

BIOSKILL

Figure 3: Elongation.

© 2002 Nature Publishing Group

Many copies of RNA can be transcribedat one time. Here, an electronmicrograph shows RNA branching likeleaf veins off the central spoke of DNA.The strand to the left has numerousfilamentous protrusions, whichrepresent the transcription of numerouscopies of RNA. The other two DNAstrands lack the filamentous protrusionsand are not being transcribed.

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 theDNA template strand. The base pairing during transcription is the same as inDNA base pairing, except that RNA contains uracil instead of thymine.Therefore, RNA polymerase uses the nucleotides CTP, GTP, ATP, and UTPto create the transcript. RNA polymerase catalyzes the formation ofphosphodiester bonds between these monomers as the transcript is created,at a rate of approximately 40 nucleotides per second. As transcriptioncontinues along the DNA, the RNA transcript separates from the DNAtemplate and the DNA double helix is re-formed (Figure 4).

A single gene may produce many RNA transcripts at the same time. Onceone RNA polymerase molecule begins the elongation phase, initiation mayoccur with another RNA polymerase molecule. By having many copies ofRNA created at the same time, the cell is capable of generating a largeamount of RNA from a single gene very quickly.

Dragon, F., et al. A large nucleolar U3ribonucleoprotein required for 18Sribosomal RNA biogenesis. Nature417, 967-970 (2002)doi:10.1038/nature00769. Used withpermission.

© 2014 Nature Education All rights reserved. Transcript

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Figure 4: The transcription process.Test your understanding of how transcription works.

Termination occurs once RNA polymerase reaches a specific sequence onthe DNA template. In bacteria, one type of terminator sequence codes for astretch of RNA that, once transcribed, creates a hairpin loop by folding backon itself. The short hairpin is created by base pairing of complementary G-Cbases within the RNA. The region downstream of the hairpin is rich in Abases in the DNA — and therefore U bases in the RNA. The formation of thestronger G-C base pairs in the hairpin, combined with the weaker U-A basepairing between RNA and DNA in the downstream region, disrupts theassociation between the RNA polymerase, the DNA template, and the RNAtranscript.

In eukaryotes, termination occurs when a sequence called a polyadenylationsignal (AAUAAA) is transcribed. Once RNA polymerase reaches 10-35 basepairs downstream of the polyadenylation signal, the RNA transcript isreleased from RNA polymerase.

Test Yourself

Describe the three phases of transcription.

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Figure 5: RNA Splicing.

© 2012 Nature Education All rightsreserved.

In eukaryotes, before translation canoccur, introns must be removed and theexons combined to form mature mRNA.

Test Yourself

Which phases of transcription could be affected by changes to DNA sequences?

In bacteria, the RNA is ready for translation as soon as it is transcribed.However, in eukaryotes the mRNA must undergo processing in the nucleusbefore 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 first20–40 nucleotides of the pre-mRNA are synthesized during transcriptionelongation, a modified guanine (G) nucleotide is added to the 5′ end of thetranscript, creating the 5′ cap. The 5′ cap helps the transcript bind to theribosome for translation. It also helps protect the mRNA from enzymaticdegradation 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 theexport of the mRNA to the cytoplasm for translation, and, like the 5′ cap, thepoly(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? Theanswer lies in a process known as RNA splicing. In eukaryotes, largeportions of the pre-mRNA molecule are removed before the mRNA isexported from the nucleus. The segments of the pre-mRNA that are includedin the final mRNA molecule are called exons. The non-coding segments thatare removed are called introns (Figure 5). There is a consensus sequence atthe junctions between exons and introns. These short sequences of DNAhave little variation between different genes.

Once the pre-mRNA is transcribed, several small nuclear ribonucleoproteinparticles (snRNPs) bind to the consensus sequences. Other proteins alsoassociate to form an RNA-protein complex known as a spliceosome. A

Figure 6: snRNPs combine to form the spliceosome, which facilitatesRNA splicing.


Small nuclear ribonucleoproteins (snRNPs) are complexes of RNA andprotein that bind to a pre-mRNA to be spliced. The pre-mRNA containsthree sites critical to the splicing process: the 5′ splice site, the 3′ splicesite, and the branch point, which usually includes an adenine base. In thefirst step, snRNPs bind to the 5′ splice site and branch point. The snRNPsare brought together, allowing the branch point to cut the 5′ splice sitefrom the adjacent exon. Next, three more snRNPs bind to the pre-mRNA,forming the complete spliceosome. The spliceosome folds the intron into alooped structure called a lariat. In the last step, the spliceosome brings theexons together and covalently links them. At the same time, the lariat ofintronic RNA is released.

spliceosome is created at each exon-intron junction. The spliceosome cutsthe pre-mRNA, removes the intron and joins the exons together (Figure 6). Inthis way, all of the introns are removed, and exons are spliced together toform the mature mRNA that is ready for translation.

Most introns do not have a known specific function, though some containregulatory sequences that affect gene expression. One effect of RNA splicingis the ability to change which sequences are treated as exons and thereforecreate different mature mRNA molecules from the same gene. This is knownas alternative RNA splicing.


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Proteins are made up of structural and functional regions called domains.For example, one domain of an enzyme may contain an active site whileanother may contain an allosteric site. In many cases, different domains arecoded for by different exons. By using alternative splicing, the same gene isable 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.

Transcription versus DNA Replication

Summary

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How Did We Discover Transcription?

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scientists do with these cells and theirincredible potential?

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cutting-edge research lead to preventionand treatment strategies that could makecancer obsolete?

Scientists are combining biology andengineering to change the world.

PRIMARY LITERATURE

Master gene KLF14 controlsgenes in metabolic syndromeIdentification of an imprinted master transregulator at the KLF14 locus related tomultiple metabolic phenotypes.

Interfering with microRNAs tocontrol gene expressionSilencing of microRNA families byseed-targeting tiny LNAs.

Classic paper: How scientistsdiscovered the enzyme that turnsRNA into DNA (1970)RNA-dependent DNA polymerase in virionsof RNA tumour viruses.

SCIENCE ON THE WEB

A collection of articles from NaturePublishing Group about DNA and howscientists study its form and function

A collection of research papers coveringseminal discoveries about transcription


49 Transcription

OBJECTIVE Explain the processes that occur during the three phases oftranscription.

During initiation, RNA polymerase and additional factors bind to the DNA andunwind it to access the template strand of DNA. During elongation, RNApolymerase adds nucleotide monomers to the growing RNA strand.Termination involves the disruption of the association between RNApolymerase, the DNA template and the RNA transcript.

OBJECTIVE Describe the molecular factors that aid in transcription.RNA polymerase catalyzes the polymerization reaction that adds nucleotidemonomers to the growing RNA molecule. Additional factors are required forthe initiation of transcription, including the sigma protein in bacteria and avariety of transcription factors, in eukaryotes.

OBJECTIVE Relate the importance of specific sequences on the DNAmolecule to the process of transcription.

Specific sequences in the DNA, such as the core promoter elements andenhancers, help bring RNA polymerase to the transcription start site.Termination is also signaled by specific sequences in the DNA that result inthe formation of a hairpin loop that disrupts the association between the DNAtemplate and the RNA transcript.

OBJECTIVE Describe the differences between eukaryotic and prokaryotictranscription.

Prokaryotes and eukaryotes use different promoter sequences and additionalfactors to initiate transcription. Different termination sequences andmechanisms are also used. In prokaryotes, the RNA transcript is ready fortranslation as soon as it is created. In eukaryotes, RNA processing mustoccur before the RNA transcript is exported from the nucleus and is ready fortranslation.

OBJECTIVE Describe RNA processing.In eukaryotes, a 5′ cap and a 3′ poly(A) tail are added to the pre-mRNA. Theintrons are spliced out to create a mature mRNA molecule that contains onlyexons. The mature mRNA is now ready to be exported from the nucleus.

elongationThe phase of transcription in which RNA polymerase moves along the DNAtemplate and incorporates nucleotides into the growing RNA transcript.

initiationThe phase of transcription in which RNA polymerase binds to the promoter of agene and begins RNA synthesis.

intronA segment of mRNA that is removed prior to translation.

promoterThe DNA sequence at which RNA polymerase binds to initiate transcription of agene.

RNA polymeraseAn enzyme that uses a DNA template to synthesize a complementary RNAmolecule during transcription.

Summary

Key Terms


TATA boxAn element of the core promoter in eukaryotic genes; contains the consensussequence TATAAA.

terminationThe phase of transcription in which RNA polymerase releases the RNA transcriptand detaches from the DNA template.

transcription factorA protein that regulates the transcription of specific genes.

transcription initiation complexThe combination of transcription factors and RNA polymerase that assembles atthe promoter of a gene prior to transcription initiation.

transcription start siteThe site at which the first RNA nucleotide is added; also known as the +1 site.


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49 Transcription

1.

5′ capsigmaTFIIBa polyadenylation signalTATA box

Which specific sequence is important for termination in eukaryotes?

2.

The holoenzyme binds the promoter.Termination sequence signals cause the RNA to be released.RNA splicingUracil is used instead of thymine.RNA polymerase adds monomers to the growing RNA transcript.

Which of the following does NOT occur during transcription in bacteria?

3.

RNA splicingTATA boxsigmaRNA polymerase IIIspliceosomes

Which of the following is important for transcription in prokaryotes?

4.

5′-AGATCCTGA-3′5′-AGAUCCUGA-3′5′-UCAGGAUCU-3′5′-TCAGGATCT-3′5′-UCAGGATCT-3′

If the template strand of DNA has the sequence 3′-TCTAGGACT-5′, what will thesequence of the transcribed RNA be?

5.

enhancerssigmapromotersa variety of transcription factorsactivators

Other than the core RNA polymerase, what proteins are required for the initiation oftranscription in bacteria?

6.

Polyadenylation signalTFIIB recognition elementinitiator elementdownstream core promoter elementTATA box

Mutation of which of these sequences would have no effect on the initiation oftranscription?

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7.

5′-CGUGUCTTCTGU-3′5′-CGAGACUUCUGA-3′3′-GCTCTGAAGACT-5′3′-GCUCUGAAGACU-5′5′-CGAGACTTCTGA-3′

If the coding strand of DNA has the sequence 5′-CGAGACTTCTGA-3′, what willthe sequence of the transcribed RNA be?

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Principles of Biology contents 49 Transcription17) 49-Transcription.pdf · Principles of Biology contents page 252 of 989 3 pages left in this module 49 Transcription Transcription