Gene Expression: Transcription

台大農藝系 遺傳學 601 20000 Chapter 5 slide 2

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.
The Transcription Process
RNA Synthesis

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).
Fig. 5.1 Transcription process
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.
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.
Fig. 5.3 Promoter, RNA-coding sequence, and terminator regions of a gene
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.
Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of
transcription
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.
 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. 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
Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA
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.
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 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 precursor-mRNA (pre-mRNA). The pre-
mRNA will be modified to produce a mature mRNA.
2. 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.
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
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.
Fig. 5.7 Assembly of the transcription initiation machinery
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.
Eukaryotic Transcription (DNA RNA) is more complex,
slower and more flexible than in Prokaryotes:
• Gross differences in prokaryotic and eukaryotic transcription.
• 5 Eukaryotic RNA Polymerase subtypes
• Core promoters on eukaryotic DNA
• Transcription factors are required for RNA Polymerase-DNA
binding/transcription
• Relative RNA content in eukaryotic cells
• rRNA production by Polymerase I and processing
• mRNA production by RNApolymerase II and processing
• tRNA production by RNApolymerase III and processing
• Removal of introns from pre-mRNA in the nucleus
Relative to prokaryotes, eukaryotes have much greater flexibility in terms
of their ability to modify protein production. This comes at the cost of
greater complexity and time needed to make a protein.
Eukaryotes have 5 different RNA polymerases
Each subtypes makes a specific type of RNA
1) RNAP I: makes rRNA in the nucleolus (darkest part)
2) RNAP II: makes mRNA and snRNA in nucleoplasm
3) RNAP III: makes rRNA and tRNA in nucleoplasm
• RNAPolymerases are massive complexes: 500,000mw
• Similar to Prokaryotic RNAPolymerases
4) Mitochondria: has mRNAP
5) Chloroplast: has cRNAP
How much of each type of RNA are contained in a cell?
rRNA: about 75%
tRNA: about 15%
mRNA: less than 10%