Transcript CHAPTER 5 Gene Expression: Transcription

Peter J. Russell
A molecular Approach 2nd Edition
CHAPTER 5
Gene Expression: Transcription
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 5 slide 1
Gene Expression: An Overview
1. Francis Crick (1956) named the flow of information from DNA 
RNAprotein the Central Dogma.
2. Synthesis of an RNA molecule using a DNA template is called
transcription. Only one of the DNA strands is transcribed. The enzyme
used is RNA polymerase.
3. There are four major types of RNA molecules:
a. Messenger RNA (mRNA) encodes the amino acid sequence of a
polypeptide.
b. Transfer RNA (tRNA) brings amino acids to ribosomes during
translation.
c. Ribosomal RNA (rRNA) combines with proteins to form a ribosome,
the catalyst for translation.
d. Small nuclear RNA (snRNA) combines with proteins to form
complexes used in eukaryotic RNA processing.
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Chapter 5 slide 2
The Transcription Process
RNA Synthesis
Animation: RNA Biosynthesis
1. Transcription, or gene expression, is regulated by
gene regulatory elements associated with each
gene.
2. DNA unwinds in the region next to the gene, due
to RNA polymerase in prokaryotes and other
proteins in eukaryotes. In both, RNA polymerase
catalyzes transcription (Figure 5.1).
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Chapter 5 slide 3
Fig. 5.1 Transcription process
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Chapter 5 slide 4
3. RNA is transcribed 5’-to-3’. The template DNA
strand is read 3’-to-5’. Its complementary DNA,
the nontemplate strand, has the same polarity as
the RNA.
4. RNA polymerization is similar to DNA synthesis
(Figure 5.2), except:
a. The precursors are NTPs (not dNTPs).
b. No primer is needed to initiate synthesis.
d. Uracil is inserted instead of thymine.
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Chapter 5 slide 5
Fig. 5.2 Chemical reaction involved in the RNA polymerase-catalyzed synthesis of
RNA on a DNA template strand
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Chapter 5 slide 6
The Transcription Process
Initiation of Transcription at Promoters
1. Transcription is divided into three steps for both prokaryotes and
eukaryotes. They are initiation, elongation and termination. The process
of elongation is highly conserved between prokaryotes and eukaryotes,
but initiation and termination are somewhat different.
2. This section is about initiation of transcription in prokaryotes. E. coli is
the model organism. (Figure 5.3)
3. A prokaryotic gene is a DNA sequence in the chromosome. The gene
has three regions, each with a function in transcription:
a. A promoter sequence that attracts RNA polymerase to begin
transcription at a site specified by the promoter.
b. The transcribed sequence, called the RNA-coding sequence. The
sequence of this DNA corresponds with the RNA sequence of the
transcript.
c. A terminator region downstream of the RNA-coding sequence that
specifies where transcription will stop.
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Chapter 5 slide 7
Fig. 5.3 Promoter, RNA-coding sequence, and terminator regions of a gene
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Chapter 5 slide 8
4. Promoters in E. coli generally involve two DNA sequences, centered at 35 bp and -10 bp upstream from the +1 start site of transcription.
5. The common E. coli promoter that is used for most transcription has
these consensus sequences:
a. For the -35 region the consensus is 5’-TTGACA-3’.
b. For the -10 region (previously known as a Pribnow box), the consensus
is 5’-TATAAT-3’.
6. Transcription initiation requires the RNA polymerase holoenzyme to
bind to the promoter DNA sequence. Holoenzyme consists of:
a. Core enzyme of RNA polymerase, containing four polypeptides (two α,
one β and one β’).
b. Sigma factor (σ).
7. Sigma factor binds the core enzyme, and confers ability to recognize
promoters and initiate RNA synthesis. Without sigma, the core enzyme
randomly binds DNA but does not transcribe it efficiently.
8. RNA polymerase holoenzyme binds promoter in two steps (Figure 5.4):
a. First, it loosely binds to the -35 sequence.
b. Second, it binds tightly to the -10 sequence, untwisting about 17 bp of
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Chapter 5 slide 9
DNA at the site, and in position to begin
transcription.
Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of
transcription
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Chapter 5 slide 10
9. Promoters often deviate from consensus. The associated genes will
show different levels of transcription, corresponding with sigma’s
ability to recognize their sequences.
10. E. coli has several sigma factors with important roles in gene
regulation. Each sigma can bind a molecule of core RNA polymerase
and guide its choice of genes to transcribe.
11. Most E. coli genes have a σ70 promoter, and σ70 is usually the most
abundant sigma factor in the cell. Other sigma factors may be produced
in response to changing conditions. Examples of E. coli sigma factors:
a. σ70 recognizes the sequence TTGACA at -35, and TATAAT at -10.
b. σ32 recognizes the sequence CCCCC at -39 and TATAAATA at -15.
Sigma32 arises in response to heat shock and other forms of stress.
c. σ54 recognizes the sequence GTGGC at -26 and TTGCA at -14. Sigma54
arises in the response to heat shock and other forms of stress.
d. σ23 recognizes the sequence TATAATA at position -15. Sigma23 is
present in cells infected with phage T4.
12. E. coli has additional sigma factors. Other bacterial species also have
multiple sigma factors.
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Chapter 5 slide 11
The Transcription Process
Elongation and Termination of an RNA Chain
1. Once initiation is completed, RNA synthesis begins. After 8–9 NTPs have been joined in
the growing RNA chain, sigma factor is released and reused for other initiations. Core
enzyme completes the transcript (Figure 5.4).
2. Core enzyme untwists DNA helix locally, allowing a small region to denature. Newly
synthesized RNA forms an RNA-DNA hybrid, but most of the transcript is displaced as
the DNA helix reforms. The chain grows at 30–50 nt/second.
3. RNA polymerase has two types of proofreading:
a. Similar to DNA polymerase editing, newly inserted nucleotide is removed by reversing
synthesis reaction.
b. b. Enzyme moves back one or more nucleotides, cleaves RNA, then resumes synthesis in
forward direction.
4. Terminator sequences are used to end transcription. In prokaryotes there are two types:
a. Rho-independent (ρ-independent) or type I terminators have two-fold symmetry that would
allow a hairpin loop to form (Figure 5.5). The palindrome is followed by 4-8U residues in the
trasncript, and together these sequences cause termination, possibly because rapid hairpin
formation destabilizes the RNA-DNA hybrid.
b. Rho-dependent (ρ-dependent) or type II terminators lack the poly(U) region, and many also lack
the palindrome. The protein ρ is required for termination. It has two domains, one binding
RNA and the other binding ATP. ATP hydrolysis provides energy for ρ to move along the
transcript and destablize the RNA-DNA hybrid at the termination region.
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Chapter 5 slide 12
Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA
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Chapter 5 slide 13
Transcription in Eukaryotes
1. Prokaryotes contain only one RNA polymerase,
which transcribes all RNA for the cell.
2. Eukaryotes have three different polymerases,
each transcribing a different class of RNA.
Processing of transcripts is also more complex in
eukaryotes.
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Chapter 5 slide 14
Eukaryotic RNA Polymerases
1. Eukaryotes contain three different RNA polymerases:
a. RNA polymerase I, located in the nucleolus, transcribes the three
major rRNAs (28S, 18S, and 5.8S).
b. RNA polymerase II, located in the nucleoplasm, transcribes
mRNAs and some snRNAs.
c. RNA polymerase III, located in the nucleoplasm, transcribes
tRNAs, 5S rRNA, and the remaining snRNAs.
2. Eukaryotic RNA polymerases are harder to study than the
prokaryotic counterpart, because they are present at low
concentrations.
3. All known eukaryotic RNA polymerases have multiple
subunits. An example is yeast RNA pol II with 12
subunits, 5 of which are also in its RNA pol III (Figure
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Chapter 5 slide 15
5.6).
Transcription of Protein-Coding Genes by RNA
Polymerase II
1. When protein-coding genes are first transcribed by RNA pol II, the product is a precursormRNA (pre-mRNA). The pre-mRNA will be modified to produce a mature mRNA.
2. Promoters for protein-coding genes are analyzed in two ways:
a. Directed mutation.
b. Comparison of sequences from known genes.
3. Results of promoter analysis reveal two types of elements:
a. Core promoter elements are located near the transcription start site and specify where
transcription begins. Examples include:
i. The initiator element (Inr), a pyramidine-rich sequence that spans the transcription
start site.
ii. The TATA box (also known as a TATA element or Goldberg-Hogness box) at -30; its
full sequence is TATAAAA. This element aids in local DNA denaturation, and sets the
start point for transcription.
b. Promoter proximal elements are required for high levels of transcription. They are
further upstream from the start site, at positions between -50 and -200. These elements
generally function in either orientation. Examples include:
i. The CAAT box, located at about -75.
ii. The GC box, consensus sequence GGGCGG, located at about -90.
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Chapter 5 slide 16
4. Various combinations of core and proximal elements are found near different genes.
Promoter proximal elements are key to gene expression.
a. Activators, proteins important in transcription regulation, are recognized by promoter
proximal elements.
b. Housekeeping (used in all cell types for basic cellular functions) genes have common
promoter proximal elements and are recognized by activator proteins found in all cells.
Examples:
i. Actin
ii.Glucose-6-phosphate dehydrogenase
c. Genes expressed only in some cell types or at particular times have promoter proximal
elements recognized by activator proteins found only in specific cell types or times.
5. Enhancers are another cis-acting element. They are required for maximal
transcription of a gene.
a. Enhancers are usually upstream of the transcription initiation site, but may also be
downstream. They may modulate from a distance of thousands of base pairs away from
the initiation site.
b. Enhancers contain short sequence elements, some similar to promoter sequences.
c. Activators bind these sequences and other protein complexes form, bringing the enhancer
complex close to the promoter and increasing transcription.
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Chapter 5 slide 17
Fig. 5.7 Assembly of the transcription initiation machinery
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Chapter 5 slide 18
6. Transcription initiation requires assembly of RNA polymerase II and binding of
general transcription factors (GTFs) on the core promoter.
a. GTFs are needed for initiation by all three RNA polymerases.
b. GTFs are numbered to match their corresponding RNA polymerase, and
lettered in the order of discovery (e.g., TFIID was the fourth GTF discovered
that works with RNA polymerase II).
7. Sequence of binding is not completely understood.
a. Binding of GTFs and RNA pol II occurs in a set order in in vitro experiments
(Figure 5.7) to produce the complete transcription initiation complex or
preinitiation complex (PIC):
b. The situation is less clear in vivo. Some data indicate that initiation complex
forms before binding promoter.
c. Transcription for eukaryotes is also complicated by the nucleosome
organization of chromosomes.
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Chapter 5 slide 19
iActivity: Investigating Transcription in BetaThalassemia Patients
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Chapter 5 slide 20
Box Fig. 5.1 Sucrose density gradient centrifugation technique for separating and
isolating RNA molecules in a mixture
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Chapter 5 slide 21
Eukaryotic mRNAs
Animation: mRNA Production in Eukaryotes
1. Eukaryotic mRNAs have 3 main parts (Figure 5.8):
a. The 5’ leader sequence, or 5’ untranslated region (5’ UTR),
varies in length.
b. The coding sequence, which specifies the amino acid sequence
of the protein that will be produced during translation. It varies
in length according to the size of the protein that it encodes.
c. The trailer sequence, or 3’ untranslated region (3’ UTR) also
varies in length and contains information influencing the
stability of the mRNA.
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Chapter 5 slide 22
Fig. 5.8 General structure of mRNA found in both prokaryotic and eukaryotic cells
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Chapter 5 slide 23
2. Eukaryotes and prokaryotes produce mRNAs
somewhat differently (Figure 5.9).
a. Prokaryotes use the RNA transcript as mRNA without
modification. Transcription and translation are coupled
in the cytoplasm. Messages may be polycistronic.
b. Eukaryotes modify pre-RNA into mRNA by RNA
processing. The processed mRNA migrates from
nucleus to cytoplasm before translation. Messages are
always monocistronic.
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Chapter 5 slide 24
Fig. 5.9 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
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Chapter 5 slide 25
Production of Mature mRNA in Eukaryotes
1. Eukaryotic pre-RNAs often have introns
(intervening sequences) between the exons
(expressed sequences) that are removed during
RNA processing. Introns were discovered in 1977
by Richard Roberts, Philip Sharp and Susan
Berger.
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Chapter 5 slide 26
5’ and 3’ Modifications
1. The newly made 5’ end of the mRNA is modified
by 5’ capping. A capping enzyme adds a guanine,
usually 7-methyl guanosine (m7G), to the 5’ end
using a 5’-to-5’ linkage. Sugars of the 2 adjacent
nt are also methylated. The cap is used for
ribosome binding to the mRNA during translation
initiation. (Figure 5.10)
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Chapter 5 slide 27
Fig. 5.10 Cap structure at the 5 end of a eukaryotic mRNA
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Chapter 5 slide 28
2. The 3’ end of mRNA is marked by a poly(A) tail (Figure
5.11).
a. Transcription of mRNA continues through the poly(A)
consensus sequence (AAUAAA).
b. Proteins bind and cleave RNA. These include:
i. CPSF (cleavage and polyadenylation specificity factor).
ii. CstF (cleavage stimulation factor).
iii. Two cleavage factor proteins (CFI and CFII).
c. After cleavage, the enzyme poly(A) polymerase (PAP) adds A
nucleotides to the 3’ end of the RNA, using ATP as a substrate.
PAP is bound to CPSF during this process.
d. PABII (poly(A) binding protein II) binds the poly(A) tail as it is
produced.
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Chapter 5 slide 29
Fig. 5.11 Diagram of the 3 end formation of mRNA and the addition of the poly(A) tail
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Chapter 5 slide 30
Introns
1. Removal of introns is necessary for mRNA maturation, as
hnRNA (Heteronuclear RNA) becomes functional
mRNA.
2. in Philip leder’s lab (1978) it was shown that the mouse
β-globin pre-mRNA (part of the cell’s hnRNA) is colinear
with the gene that encodes it, while the mature β-globin
mRNA is shorter than the gene. The missing RNA was an
intron that was removed during RNA processing.
3. Introns are found in most eukaryotic genes that encode
proteins, and have also been found in bacteriophage
genes.
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Chapter 5 slide 31
Processing of Pre-mRNA to Mature mRNA
Animation: RNA Splicing
1. Events in eukaryotic mRNA production are
summarized in Figure 5.12. They include:
a. Transcription of the gene by RNA polymerase II.
b. Addition of the 5’ cap.
c. Addition of the poly(A) tail.
d. Splicing to remove introns.
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Chapter 5 slide 32
Fig. 5.12 General sequence of steps in the formation of eukaryotic mRNA
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Chapter 5 slide 33
2. Introns typically begin with 5’-GU, and end with AG-3’, but
mRNA splicing signals involve more than just these two
small sequences.
a. Spliceosomes are small nuclear ribonucleoprotein particles
(snRNPs) associated with pre-mRNAs.
b. Spliceosome principal snRNAs are U1, U2, U4, U5, and
U6.
i. Each snRNA is associated with several proteins.
ii. U4 and U6 are part of the same snRNP. Others are in
their own snRNPs.
iii. Each snRNP type is abundant (≧105 copies per nucleus).
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Chapter 5 slide 34
3. The steps of splicing are outlined in Figure 5.13:
a. U1 snRNP binds the 5’ splice junction of the intron, as a result of base-pairing
of the U1 snRNA to the intron RNA.
b. U2 snRNP binds the branch-point sequence upstream of the 3’ splice junction.
c. U4/U6 and U5 snRNPs interact, then bind the U1 and U2 snRNPs, creating a
loop in the intron.
d. U4 snRNP dissociates from the complex, forming the active spliceosome.
e. The spliceosome cleaves the intron at the 5’ splice junction, freeing it from
exon 1. The free 5’ end of the intron bonds to a specific nucleotide (usually A)
in the branch-point sequence to form an RNA lariat.
f. The spliceosome cleaves the intron at the 3’ junction, liberating the intron
lariat. Exons 1 and 2 are ligated, and the snRNPs are released.
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Chapter 5 slide 35
Fig. 5.13 Model for intron removal by the spliceosome
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Chapter 5 slide 36
Self-Splicing of Introns
1. The rRNA genes of most species do not contain introns.
2. Some species of the protozoan Tetrahymena have a 413-bp
intron in their 28S rRNA sequence. Tom Cech (1982) showed
that splicing of this intron (called a group I intron) is proteinindependent. The intron self-splices by folding into a
secondary structure that catalyzes its own excision.
3. Group I introns are rare, but examples occur in:
a. Nuclear rRNA genes.
b. Some mitochondrial mRNA genes.
c. Some tRNA and mRNA genes in bacteriophages.
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Chapter 5 slide 37
4. The steps in self-splicing of a group I intron in
Tetrahymena are shown in Figure 5.14:
a. The pre-rRNA is cleaved at the 5’ splice junction and guanosine is
added to the 5’ end of the intron.
b. The intron is cleaved at the 3¢ splice junction.
c. The two exons are joined together.
d. The excised intron forms a lariat structure, which is cleaved to
produce a circular RNA and a short linear piece of RNA.
5. Self-splicing is not an enzyme activity, because the RNA
is not regenerated in its original form at the end of the
reaction. However, the discovery of ribozymes (catalytic
RNAs) has significantly altered our view of the
biochemistry involved in the origin of life.
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Chapter 5 slide 38
Fig. 5.14 Self-splicing reaction for the group I intron in Tetrahymena pre-rRNA
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Chapter 5 slide 39
RNA Editing
1. RNA editing adds or deletes nucleotides from a pre-mRNA, or
chemically alters the bases, resulting in an mRNA with bases that don’t
match its DNA coding sequence.
2. Examples have been found in a number of organisms: (Figure 5.15)
a. In Trypanosome brucei (a protozoan causing sleeping sickness) the
cytochrome oxidase subunit III gene (from mitochondrial DNA) does
not match its mRNA. Uracil residues have been added and removed,
and over 50% of the mature mRNA consists of posttranscriptionally
added Us. This RNA editing is mediated by a guide RNA (gRNA) that
pairs with the mRNA, cleaving it, adding the Us and ligating it.
b. In Physarum polycephalum, a slime mold, single C nucleotides are
added posttranscriptionally at many positions in mRNAs from several
mitochondrial genes.
c. In higher plants, many mitochondrial and chloroplast mRNAs undergo
C-to-U editing, including production of an AUG initiation codon from
an ACG codon in some chloroplast mRNAs.
d. In mammals, C-to-U editing occurs in the mRNA for apolipoprotein B,
resulting in a tissue-specific stop codon. A-to-G editing occurs in the
glutamate receptor mRNA, and pyrimidine editing occurs in some
tRNAs.
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Chapter 5 slide 40
Fig. 5.15 Comparison of the DNA sequences of the cytochrome oxidase subunit III
gene in the protozoans Trypanosome brucei (TB), Crithridia fasiculata (Cf),
and Leishmania tarentolae (Lt), aligned with the conserved mRNA for Tb
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Chapter 5 slide 41
Transcription of Other Genes
1. Genes that do not encode proteins are also
transcribed, including genes for rRNA, tRNA and
snRNA.
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Chapter 5 slide 42
Ribosomal RNA and Ribosomes
1. Ribosomes are the catalyst for protein synthesis,
facilitating binding of charged tRNAs to the
mRNA so that peptide bonds can form. A cell
contains thousands of ribosomes.
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Chapter 5 slide 43
Ribosome Structure
1. Ribosomes in both prokaryotes and eukaryotes consist of two subunits
of unequal size (large and small), each with at least one rRNA and
many ribosomal proteins.
2. E. coli is the model for a prokaryotic ribosome. It is 70S, with 50S and
30S subunits (Figure 5.16).
a. The 50S subunit contains the 23S rRNA (2,904 nt) and 5S rRNA (120
nt), plus 34 different proteins.
b. The 30S subunit contains the 16S rRNA (1,542 nt), plus 20 different
proteins.
3. Eukaryotic ribosomes are larger and more complex than prokaryotic
ones, and vary in size and composition among organisms. Mammalian
ribosomes are an example; they are 80S, with 60S and 40S subunits
(Figure 5.16).
a. The 60S subunit contains the 28S rRNA (~4,700 nt), the 5.8S rRNA
(156 nt) and the 5S rRNA (120 nt), plus about 50 proteins.
b. The 40S subunit contains the 18S rRNA (~1,900 nt) plus about 35
proteins.
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Chapter 5 slide 44
Fig. 5.16 Model of the complete (70S) ribosome of E. coli
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Chapter 5 slide 45
Fig. 5.17 Composition of whole ribosomes and of ribosomal subunits in mammalian
cells
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Chapter 5 slide 46
Transcription of rRNA Genes
1. DNA regions that encode rRNA are called ribosomal
DNA (rDNA) or rRNA transcription units.
2. E. coli is a typical prokaryote, with seven rRNA coding
regions, designated rrn, scattered in its chromosome
(Figure 5.18).
a. Each rrn contains the rRNA genes 16S-23S-5S, in that order,
with tRNA sequences in the spacers.
b. A single pre-rRNA transcript is produced from rrn, and cleaved
by RNases to release the rRNAs. Cleavage occurs in a complex
of rRNA and ribosomal proteins, resulting in functional
ribosomal subunits.
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Chapter 5 slide 47
Fig. 5.18 rRNA genes and rRNA production in E. coli
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Chapter 5 slide 48
3. Eukaryotes generally have many copies of the rRNA genes (Figure 5.19).
a. The three rRNA genes with homology to prokaryotic rRNA genes are 18S-5.8S28S, in that order. In the chromosome these genes are tandemly repeated 100–
1,000 times to form rDNA repeat units. The 5S rRNA gene copies are located
elsewhere in the genome.
b. A nucleolus forms around each rDNA repeat unit, and then they fuse to make
one nucleolus. Ribosomal subunits are produced in this structure by addition of
the 5S rRNA and ribosomal proteins.
c. RNA polymerase I transcribes the rDNA repeat units, producing a pre-rRNA
molecule containing the 18S, 5.8S and 28S rRNAs, separated by spacer
sequences.
i. External transcribed sequences (ETS) located upstream of 18S and
downstream of 28S.
ii. Internal transcribed spacers (ITS) located on each side of 5.8S.
iii. Nontranscribed spacer (NTS) sequence is between each rDNA repeat unit,
and includes the promoter.
4. Specific cleavage steps free the rRNAs from their transcript as part of pre-rRNA
processing that takes place in the complex of pre-rRNA, 5S rRNA, and ribosomal
proteins. The result is formation of 40S and 60S ribosomal subunits, which are then
transported to the cytoplasm.
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Chapter 5 slide 49
Fig. 5.19 rRNA genes and rRNA production in eukaryotes
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Chapter 5 slide 50
Transcription of Genes by RNA Polymerase III
1. Genes transcribed by RNA polymerase III include:
a. The eukaryotic 5S rRNA (120 nt), found in the 60S ribosomal subunit.
(This rRNA has no counterpart in the prokaryotic ribosome.)
b. The tRNAs (75-90 nt), which occur in repeated copies in the eukaryotic
genome.
i. Each tRNA has a different sequence.
ii. All tRNAs have CCA (added posttranscriptionally) at their 3’ ends.
iii. Extensive chemical modifications are performed on all tRNAs
after transcription.
iv. All tRNAs can be shown in a cloverleaf structure, with
complementary base pairing between regions to form four stems
and loops (Figure 5.20). Loop II contains the anticodon used to
recognize mRNA codons during translation. Folded tRNAs
resemble an upside-down “L”.
c. Some snRNAs (the others are transcribed by RNA polymerase II).
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Chapter 5 slide 51
Fig. 5.20 Transfer RNA (a) Cloverleaf structure of yeast alanine tRNA
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Chapter 5 slide 52
2. Promoter sequences for 5S rRNA and tRNA genes are
typically within the sequences that will be transcribed, hence
internal control regions (IRC). Promoters for the snRNA genes
transcribed by RNA pol III are typically upstream of the genes.
3. Transcription of 5S rDNA produces a mature 5S rRNA, and no
sequences need to be removed.
4. Some tRNA genes contain introns. About 10% of the 400 yeast
tRNA genes have introns. If present, introns usually are found
just 3’ to the anticodon, and they are removed by a specific
endonuclease, with splicing completed by RNA ligase.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 53