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Chapter 5
Gene Expression:
Transcription
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The flow of information from DNA RNA
protein was called the Central Dogma by
Francis Crick in 1956.
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Synthesis of an RNA molecule using a DNA
template is called transcription. The process of
coding the RNA information into proteins is
called translation.
Transcription
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Only one of the DNA strands is transcribed. The enzyme used is RNA
polymerase.
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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
ribosome, the catalyst for translation.
proteins to form a
d. Small nuclear RNA (snRNA) combines with proteins to form
complexes used in eukaryotic RNA processing
The Transcription Process
„ RNA Synthesis
1. Transcription, or gene expression, is regulated by
gene regulatory elements associated with each gene.
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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).
3. RNA is transcribed 5’-to-3’. The template DNA
strand is read 3’-to-5’. Its complementary DNA, the
non-template strand, has the same polarity as the RNA.
4. RNA polymerization is similar to DNA
synthesis (Figure 5.2), except:
ƒ a. The precursors are rNTPs (not dNTPs).
ƒ b. No primer is needed to initiate synthesis.
ƒ c. Uracil is inserted instead of thymine.
ƒ d. Only one strand is copied (coding strand
3’to5’)
Transcription, the process
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Transcription is divided into three steps for both
prokaryotes and eukaryotes. They are:
Initiation
Elongation
Termination.
The process of elongation is highly conserved
between prokaryotes and eukaryotes, but
initiation and termination are somewhat
different.
Transcription in prokaryotes
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E. coli is the model organism (Figure 5.3).
A prokaryotic gene is a DNA sequence in the chromosome.
This gene has three regions. Each of these regions has a
function in transcription:
a. The promoter: A specific sequence that attracts the RNA
polymerase to begin transcription at a specific location.
b. RNA-coding sequence. This is the DNA information that is
going to be transcribed into mRNA.
c. A terminator region that specifies where transcription will
stop.
The RNA polymerase
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RNA polymerase is a holoenzyme. Holoenzymes are enzymes that
require a cofactor binding to the Protein portion for the enzyme to be
active. The protein portion is called “the apoenzyme”.
The RNA polymerase comprises of
a. A core enzyme, containing four polypeptides
(two α, one β, and one β’).
b. Sigma factor (σ).
Sigma factor binds the core enzyme, and confers ability to recognize
promoters.
The Promoter
The presence of a promoter
sequence determines which
strand of the DNA helix is
the template and inside the
promoter we find the
starting point for the
transcription of a gene.
The promoter also includes a
binding site for RNA
polymerase (several dozen
nucleotides upstream of the
start point). The enzyme
recognizes this binding site
and binds directly to the
promoter region.
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Promoters in E. coli generally involve two DNA
sequences, centered at -35bp and -10bp
upstream from the +1 start site of transcription.
Very commonly, not always, for the -35 region
the consensus is 5’-TTGACA-3’ and for the -10
region (previously known as a Pribnow box), the
consensus is 5’-TATAAT-3’.
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Initiation
Transcription initiation requires the RNA polymerase
holoenzyme to bind to the promoter DNA sequence . It
takes places in two steps:
a. First, it loosely binds to the -35 sequence of dsDNA
(closed promoter complex).
b. Second, it binds tightly to the -10 sequence, untwisting
about 17bp of DNA at the site, and in position to begin
transcription (open promoter complex).
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At this point the RNA polymerase is correctly
oriented to begin transcription at the +1
nucleotide.
After about 10 bases have been polymerized
(transcribed) the sigma factor dissociates from
the enzyme.
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The associated genes will show different levels of
transcription, corresponding with sigma’s ability to
recognize their sequences.
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E. coli has several different sigma factors with
important roles in gene regulation. Other bacterial
species also have multiple sigma factors.
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For example, most E. coli genes have a σ70 promoter,
and σ70 is usually the most abundant sigma factor in
the cell. However, other sigma factors may be
produced in response to changing conditions.
Examples of E. coli sigma factors are: σ32, σ54 and σ23
Elongation of an RNA Chain
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1. Once initiation is completed, RNA synthesis
begins. After 8–9 rNTPs 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–
50nt/second.
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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.
Termination
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Terminator sequences are used to end
transcription. In prokaryotes there are two types:
a. Rho-independent (r-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 4–8 U residues in the
transcript, and when these sequences are
transcribed, they cause termination.
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Rho-dependent (r-dependent) or type II
terminators lack the poly(U) region, and many
also lack the palindrome. The protein rho is
required for termination. It requires energy.
Transcription in Eukaryotes
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Prokaryotes contain only one RNA polymerase,
which transcribes all RNA for the cell.
Eukaryotes have three different polymerases,
each transcribing a different class of RNA.
Processing of transcripts is also more complex
in eukaryotes.
Eukaryotic RNA Polymerases
„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.
„All known eukaryotic RNA polymerases have multiple
subunits
Initiation of transcription.
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The transcription initiation complex is composed of promoter
sequences and DNA binding proteins. These two components of
transcription are normally described as cis-acting elements
(promoter sequences) and trans-acting factors (binding
proteins)
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Cis-acting elements are- DNA sequences in the vicinity of the
structural portion of a gene that are required for gene expression.
The promoter of protein coding genes in eukaryote cells are located
about 200 base pairs upstream of the transcription initiation site
and contains various elements.
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We can divide the promoter into two areas, the core promoter and
the promoter proximal elements.
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The core promoter is usually a DNA sequence
located about -50 from the transcription starting
point. This sequence is divided in sections or
“elements”. The best known elements are the
INR, initiator and the TATA box(GoldbergHogness box). The core promoter determines
where the transcription will begin.
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The promoter proximal elements are further
up upstream (from -50 to -200) and the most
well known element is the CAAT box located
about -75 and the CG box. The promoter
proximal elements determine when and how the
gene is going to be expressed.
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Activators: Proteins that regulate transcription.
Enhancers: Required for maximal transcription
of a gene. They can be upstreqam or
downstream.
Transcription initiation factors. GTF’ that are
numbered and
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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).
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Sequence of binding is not completely
understood.
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Eukaryotic mRNAs
1. Eukaryotic mRNAs have three 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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Eukaryotes modify pre-RNA into mRNA by
RNA processing and then the processed mRNA
migrates from nucleus to cytoplasm before
translation.
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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.
5’ Modifications
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The newly made 5’ end of the mRNA is
modified by 5’ capping. The 5' cap is
modified guanine nucleotide
(methylated and known as a 7-methyl
guanosine m7G ) is added to 5‘ using a
5’-to-5’ linkage.
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Sugars of the two adjacent nt are also
methylated. This modification is critical
for recognition and proper ribosome
binding to the mRNA during
translation initiation (Figure 5.10). It
may also be important for other
essential processes, such as splicing and
transport.
The 3’ end of mRNA is marked by a
poly(A) tail (Figure 5.11).
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a. Transcription of mRNA continues through
the poly(A) sequence (AAUAAA).
b. Proteins bind and cleave RNA.
c. After cleavage, the enzyme poly(A)
polymerase (PAP) adds A nucleotides to the 3’
end of the RNA, using ATP as a substrate.
d. PABII (poly(A) binding protein II) binds the
poly(A) tail as it is produced.
RNA splicing
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Introns typically begin with 5’-GU, and end with AG3’, but more than just these sequences is needed to
specify a junction between exon and intron.
The removal of the introns is called RNA splicing and
it involves a complex called spliceosomes.
A spliceosome consists of the preRNA and a group of
proteins; the small nuclear ribonucleoprotein particles
(snRNPs) also known as snurps. There are labeled U1,
U2, U3, U4, U5 and U6.
Transcription on non-protein coding
genes.
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tRNA and rRNA’s do not code for proteins,
nevertheless their sequence needs to be
transcribed for their existence.
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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.
Prokaryote Ribosome
The prokaryotic ribosome is a 70S
ribosome, with two subunits : A large, 50S,
and the small, 30S. subunits (Figure 5.16).
The 50S subunit consists of two molecules
of rRNA, the 23S rRNA (2,904nt) and 5S
rRNA (120nt), plus 34 different proteins.
The 30S subunit contains only one rRNA
molecule, the 16S rRNA (1,542nt), plus 20
different proteins.
S or Sverberg is a
unit of
centrifugation
The eukaryotic ribosome
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 5.17).
The 60S subunit contains the 28S rRNA (~4,700nt), the 5.8S rRNA
(156nt), and the 5S rRNA (120nt), plus about 50 proteins.
The 40S subunit contains the 18S rRNA (~1,900nt) plus about
35 proteins.
Transcription of rRNA Genes
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DNA regions that encode rRNA are called
ribosomal DNA (rDNA) or rRNA transcription
units (genes?).
E. coli is a typical prokaryote, with seven rRNA
coding regions, designated rrn, scattered in its
chromosome (Figure 5.18).
Each rrn contains the rRNA genes 16S-23S-5S,
in that order, with tRNA sequences in the
spacers.
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.