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Principles of Biology
49
contents
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.
page 252 of 989
3 pages left in this module
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
contents
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?
Submit
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.
Submit
Test Yourself
Which phases of transcription could be affected by changes to DNA sequences?
Submit
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.
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 253 of 989
2 pages left in this module
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.
contents
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
contents
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