Transcript CHAPTER 5 Gene Expression: Transcription

Peter J. Russell
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.
• c. No proofreading occurs.
• 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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台大農藝系 遺傳學 601 20000
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.
• 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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台大農藝系 遺傳學 601 20000
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 DNA at
the site, and in position to begin transcription.
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Chapter 5 slide 9
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. 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
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
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, synthesizes three of the
four rRNAs found in ribosomes: three of the RNAs (the 28S, 18S, and
5.8S rRNA molecules).
• b. RNA polymerase II, located in the nucleoplasm, synthesizes
messenger RNAs (mRNAs; translated to produce polypeptides) and
some small nuclear RNAs (snRNAs), some of which are involved in
RNA processing events.
• c. RNA polymerase III, also located in the nucleoplasm, synthesizes the
transfer RNAs (tRNAs), which bring amino acids to the ribosome; 5S
rRNA, the fourth rRNA molecule found in each ribosome; and the
small nuclear RNAs (snRNAs) not made by RNA polymerase II.
• 2. Eukaryotic RNA polymerases are harder to study than the
prokaryotic counterpart, because they are present at low concentrations.
Inhibition by α-amanitin is a useful research tool, since RNA pol II is
very sensitive, RNA pol III less so and RNA pol I is relatively
insensitive.
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Chapter 5 slide 15
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
precursor-mRNA (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. Basal promoter elements are located near the transcription start site. Examples include:
• i. The TATA box (aka TATA element or Goldberg-Hogness box) at -25; its full sequence is
TATAAAA. This element aids in local DNA denaturation, and sets the start point for
transcription.
• ii. The initiator element (Inr), a pyramiding-rich sequence near the transcription start site.
•
b. Promoter proximal elements 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 basal and proximal elements are found near
different genes, and no one element is essential for transcription initiation.
•
5. Basal transcription factors (TFs) are needed for initiation by all 3 RNA
polymerases.
• a. Each TF works with only one type of RNA polymerase.
• b. TFs are numbered to match their corresponding RNA polymerase, and assigned a
letter in the order of their discovery (e.g., TFIID was the fourth TF discovered that
works with RNA polymerase II).
•
6. For protein-coding genes, binding of TFs and RNA pol II occurs in a set
order (Figure 5.5):
• a. TFIID binds the TATA box, forming an initial committed complex.
• b. TFIIB binds the TFIID-TATA box complex.
• c. The TFIID-TATA box plus TFIIB complex recruits RNA polymerase II and TFIIF,
producing the minimal transcription initiation complex.
• d. TFIIE and TFIIH bind, producing the complete transcription initiation complex,
or preinitiation complex (PIC).
•
7. The PIC allows only a low level of transcription. Higher levels are induced
by activator factors that bind DNA sequences called enhancers. Interaction
between the enhancer-activator factor complex and the PIC stimulates
transcription.
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Chapter 5 slide 17
Fig. 5.6 Events that may occur during the initiation of transcription catalyzed by RNA
polymerase II
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 18
• 8. Characteristics of enhancers:
• a. They are found in single or multiple copies.
• b. They function in either orientation.
• c. They function upstream, downstream or within the gene, although
they are usually located upstream.
• d. They may be several kb from the gene they control.
• 9. Silencers have properties similar to enhancers, except that they
decrease transcription. Repressor factors bind to them. Silencers are not
as common as enhancers.
• 10. The interaction of transcription factors binding promoters,
enhancers and silencers results in cell- and tissue-specific gene
expression.
• 11. Upstream activator sequences (UASs) in yeast are similar to
enhancers, but cannot function when located downstream of the
promoter.
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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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台大農藝系 遺傳學 601 20000
Chapter 5 slide 21
Eukaryotic mRNAs
• Animation: mRNA Production in Eukaryotes
• 1. Eukaryotic mRNAs have 3 main parts (Figure 5.7):
• 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. 5.7 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 5.8).
• 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. 5.8 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
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 (Figure 5.9).
Sugars of the 2 adjacent nt are also methylated.
The cap is used for ribosome binding to the
mRNA during translation initiation.
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Chapter 5 slide 27
Fig. 5.9 Cap structure at the 5 end of a eukaryotic mRNA
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台大農藝系 遺傳學 601 20000
Chapter 5 slide 28
• 2. The 3’ end of the pre-RNA has 50–250 adenines added enzymatically
to form a poly(A) tail. The poly(A) tail is important in mRNA stability,
and also plays a role in transcription termination, since RNA
polymerase II does not rely directly on a signal in the DNA. Mammals
are an example (Figure 5.10):
• a. Transcription of mRNA continues through the poly(A) consensus
sequence (AAUAAA), the poly(A) site and the GU-rich sequence.
• b. A protein called CPSF (cleavage and polyadenylation specificity
factor) binds the AAUAAA signal.
• c. A protein called CstF (cleavage stimulation factor) binds to the GUrich sequence.
• d. CPSF and CstF bind to each other, producing a loop in the RNA.
• e. CFI and CFII bind near the poly(A) site, and RNA is cleaved.
• f. After cleavage, the enzyme poly(A) polymerase (PAP) binds to CPSF
and adds A nucleotides to the 3’ end of the RNA, using ATP as a
substrate.
• g. PABII (poly(A) binding protein II) binds the poly(A) tail as it is
produced.
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Chapter 5 slide 29
Fig. 5.10 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 horter 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 5.11. 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.
• 2. RNA splicing requires signals so that the splicing
machinery can distinguish between introns and exons.
Introns typically begin with 5’-GU, and end with AG-3’,
but the splicing signals involve more than just these two
small sequences.
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Chapter 5 slide 32
Fig. 5.11 General sequence of steps in the formation of eukaryotic mRNA
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台大農藝系 遺傳學 601 20000
Chapter 5 slide 33
• 3. Events in splicing together two exons (designated 1 and 2) are shown
in Figure 5.12
• a. cleavage occurs at the 5’ splice junction of exon 1 and the intron.
• b. The G nucleotide at the free 5’ end of the intron joins with a specific
A nucleotide (18-38 nt upstream of the 3’ spice junction) in the branchpoint sequenc of the intron, forming an RNA lariat structure.
• i. in mammals, the branch-point consensus sequence is YNCURAY.
• ii. In yeast, the branch point consensus sequence is UACUAAC; its
position is more variable than in mammals.
• c. The bond forming the lariat is a 2’-5’ phosphodiester linkage
between the 5’ phosphate of the free guanine nt at the end of introns,
and the 2’ OH of the adenine nt in the branch-point sequence.
• d. The introns lariat is excised, and the exons are joined to form a
spliced mRNA. The intronsRNA is degraded by the cell.
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Chapter 5 slide 34
Fig. 5.12 Details of intron removal from a pre-mRNA molecule
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Chapter 5 slide 35
• 4. Splicing occurs in the nucleus, mediated by spliceosomes consisting
of small nuclear ribonucleoprotein particles (snRNPs) bound to the premRNA. The snRNPs consist of snRNAs associated with proteins.
• a. Each of the 6 principal snRNAs (named U1-U6) is associated with 610 proteins to form the snRNPs.
• b. Some of the proteins are specific to particular snRNPs, and others
are found in all snRNPs.
• c. The U4 and U6 snRNAs occur within the same snRNP (U4/U6
snRNP). All the other snRNPs have only a single snRNA.
• d. The snRNPs are abundant in the nucleus.
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Chapter 5 slide 36
• 5. 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 37
Fig. 5.13 Model for intron removal by the spliceosome
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Chapter 5 slide 38
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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:
• 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. 5.14 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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台大農藝系 遺傳學 601 20000
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 5.15).
• 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
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Chapter 5 slide 45
Fig. 5.16 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 5.17).
• 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. 5.17 rRNA genes and rRNA production in E. coli
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台大農藝系 遺傳學 601 20000
Chapter 5 slide 48
•
3. Eukaryotes generally have many copies of the rRNA genes (Figure 5.18).
•
a. The three rRNA genes with homology to prokaryotic rRNA genes are 18S-5.8S-28S, 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.
•
d. The RNA polymerase I promoter inhumans has two domains, a core promoter element
overlapping the transcription start site, and an upstream control element (UCE). Two
transcription factors bind to the promoter. (Figure 5.19)
• i. human upstream binding factor (hUBF) bind both promoter elements.
• ii. SL1 binds the complex of hUBF and DNA. SL1 consists of TBF (TATA binding
protein, which is also found in TFIID) and 3 TAFs (TBF-associated factors, fidderent
from those in TFIID).
•
4. When hUBF and SL1 have bound the promoter, the RNA polymerase I bind and
transcription begins. Transcription terminates at specific termination site downstream.
•
5. 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. 5.18 rRNA genes and rRNA production in eukaryotes
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台大農藝系 遺傳學 601 20000
Chapter 5 slide 50
Fig. 5.19 Transcription factors involved in the initiation of human rDNA transcription
by RNA polymerase I
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Chapter 5 slide 51
Self-Splicing of Introns in Tetrahymena PrerRNA
• 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 protein-independent. The intron
self-splices by folding into a secondary structure that catalyzes its own
excision.
• 3. Other group I introns occur in:
• a. The rRNA genes and some mRNA genes in mitochondria of yeast,
Neurospora and other fungi.
• b. The rRNA genes of all insect species examined.
• c. The rRNA and some mRNA and tRNA genes in bacteriophages.
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Chapter 5 slide 52
• 4. The steps in self-splicing of a group I intron in Tetrahymena are
shown in Figure 5.20:
• 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. Removal of spacers is a different activity from removal of introns,
because spacer removal releases a free rRNA that remains separate,
while removal of an intron results in ligation of the RNA sequences that
flanked the intron.
• 6. 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.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 53
Fig. 5.20 Self-splicing reaction for the group I intron in Tetrahymena pre-rRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 54
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(Figure 5.21),
with complementary base pairing between regions to form four
stems and loops. Loop II contains the anticodon used to recognize
mRNA codons during translation. Folded tRNAs resemble an
upside-down “L” (Figure 5.22).
• c. Some snRNAs (the others are transcribed by RNA polymerase II).
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Chapter 5 slide 55
Fig. 5.21 Cloverleaf structure of yeast alanine tRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 56
Fig. 5.22 Three-dimensional structure of yeast phenylalanine tRNA as determined
by X-ray diffraction of tRNA crystals
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 57
•
2. Promoter sequences for 5S rRNA and tRNA genes are typically within the sequences that
will be transcribed, hence internal control region (ICR). Promoters for the snRNA genes
transcribed by RNA pol III are typically upstream of the genes.
•
3. Transcription initiation for 5S rRNA and tRNAs requires binding of TFIIIs to the ICR,
allowing RNA polymerase III to bind.
•
•
a. The 5S rDNA has two ICR domains, boxA and boxC.
•
b. The tDNA has two ICR domains, boxA and boxB.
4. The ICRs interact with transcription factors TFIIIA, TFIIIB and TFIIIC. Formation of the
transcription complex on 5S rDNA illustrates this interaction (Figure 5.23)
•
a. TFIIIA is bound to boxC, TFIIIC can bind to boxA.
•
b. When TFIIIA is bound to boxC, TFIIIC can bind to boxA.
•
c. TFIIIB then binds to TFIIIA and TFIIIC (not to the DNA directly).
•
d. TFIIIB functions as a transcription initiation factor by positioning RNA polymerase III
correctly on the gene.
•
e. RNA polymerase III then begins transcription 50 bp upstream from boxA, at the beginning of
the gene.
•
f. Once the transcription factors are positioned on the 5S rDNA, they initiate successive rounds
of transcription without dissociating from the DNA.
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Chapter 5 slide 58
Fig. 5.23 Model for the formation of a transcription initiation complex on a 5S rDNA ICR
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 59
• 5. Transcription termination for the 5S rRNA and tRNA
genes uses simple sequences at the 3’ end of the genes.
• 6. Transcription of 5S rDNA produces a mature 5S rRNA,
and no sequences need to be removed.
• 7. Transcription of tRNA genes produces a pre-tRNA with
extra sequences at each end, and introns in the tRNAs for
certain amino acids. If present, introns are usually found
just 3’ to the anticodon, and in many cases the anticodon
pairs with the intron in the pre-tRNA. Introns are
removed by a specific endonuclease, and splicing is
completed by RNA ligase.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 60
Fig. 5.24 Cloverleaf models for yeast precursor tRNA.Tyr and mature tRNA.Tyr
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 5 slide 61