Transcription

The transcription of one strand of DNA into a
complementary RNA molecule is the first step
in gene expression. Multiple proteins (called
transcription factors) form a transcription complex,
which binds to DNA. Although there are
differences in transcription in prokaryotes and
eukaryotes, much of the basic process is the
same. Transcription is catalyzed by RNA polymerase.
RNA polymerase in E. coli has five subunits
(two !, two ", and one sigma), each encoded
by its own genes. RNA polymerases in
eukaryotes are complex (see p. 214). Eukaryotic
RNA polymerase consists of three different
enzymes, which transcribe different types of
genes.

Transcription by RNA polymerase

Transcription begins with recognition of a
specific site by RNA polymerase (1). Here the
double helix is opened and begins to unwind.
RNA synthesis begins (initiation, 2) and continues
with elongation (3). As the polymerase
moves along the DNA, mRNA is synthesized.
The DNA that has been transcribed rewinds into
the double helix behind the polymerase. At termination
(4), the RNA polymerase is removed
fromtheDNA. At this point, the formation of the
unstable primary transcript is completed. Since
it is unstable, it is immediately translated in
prokaryotes and modified (processed) in
eukaryotes (see p. 50). All the processes aremediated
by the complex interaction of a variety of
enzymes.

Polymerase binding site

Bacterial RNA polymerase binds to a specific region
of about 60 base pairs of the DNA. Several
active centers can be identified (not shown
here).

Promoter of transcription

Transcription must begin at a specific position
of DNA, just upstream (at the 5! end) of a gene.
This transcription initiation site is called a promoter.
A promoter is a short nucleotide
sequence of DNA that regulates the onset of
transcription by binding to RNA polymerase.
Two distinct promoter regions can be recognized
above the transcription starting point.
These sequences are evolutionarily highly conserved
(consensus sequences). In prokaryotes, a
promoter with a consensus sequence consisting
of six base pairs, TATAAT (also called a Pribnow
box after its discoverer) is located 10 base pairs
above the starting point; another region of conserved
sequences, TTGACA, is located 35 base
pairs above the gene (at the 5! end). These
sequences are referred to as the "10 box and the
"35 box respectively (the term “box” is derived
from the sequence identity or similarity in all
genes). In eukaryotes, the location and the
sequences of the promoters differ slightly from
those of the prokaryotes

A transcription unit

A transcription unit is all of the DNA sequences
in a given segment that are used in transcription.
It begins at the promoter and ends at the
terminator. The region around the promoter at
the 5! end is designated proximal; that around
the terminator at the 3! end is designated distal.

Determination of the starting point of transcription

One way to identify an active gene is to determine
the starting point of transcription. This
can be done by comparing the RNA formed and
the DNA template. After transcription, the RNA
formed (single-stranded) is hybridized to a
complementary single strand of DNA (RNA/
DNA hybridization). An endonuclease (S1 nuclease)
that cleaves only single-stranded DNA
degrades the nonhybridized single strand of
DNA, while the hybridized strand is protected
(RNA protection assay). Subsequently, the RNA
can be removed and the transcribed DNA segment
analyzed (e.g., its size or sequence determined).

Control of Gene Expression in Bacteria by Induction

The regulation of gene expression is a basic
function of prokaryotic and eukaryotic organisms.
Prokaryotic organisms rely entirely on
their ability to adapt rapidly to changes in external
conditions. Substances usually present in
the nutrient medium need not be synthesized
by the bacterium itself. On the other hand, substances
not present must be synthesized by the
cell. The control of gene expression occurs at
different levels. Regulator proteins may act as
repressors (suppressing RNA polymerase activity)
or as activators (inducing RNA polymerase
activity). Control of prokaryotic genes is often
facilitated in that functionally related genes
usually lie together and therefore can be regulated
together

Induction of enzymes in bacteria

The presence of certain substances in the
nutrient medium induces the synthesis of
enzymes for their utilization in bacteria. An example
in Escherichia coli (E. coli) is the activation
of three enzymes for lactose catabolism by
lactose. Within 10 minutes after lactose is
added to the nutrient medium, the enzymes !-
galactosidase, !-galactoside permease, and !-
galactoside transacetylase increase manyfold
(1). !-Galactosidase is the enzyme that splits
lactose into galactose and glucose (2).

The lactose operon in E. coli

A series of genes whose regulation is coordinated
is called an operon. Three structural
genes that code for the synthesis of lactose-degrading
enzymes (genes lacZ, lacY, and lacA)
form the lactose operon (lac operon). These
three genes are regulated by a promoter at the
5! end and are transcribed into a common
mRNA (polycistronic transcipt). Normally they
show little activity because a lac repressor inhibits
(laci) lac mRNA snythesis. The former is
the gene product of the lacI regulator gene. E.
coli can use lactose as its sole source of carbon
and energy because large amounts of !-galactosidase
can be synthesizedwithin a short time.

Control of the lac operon

The three structural genes of the lac operon,
lacZ, lacY, and lacA, are controlled bymeans of a
repressor protein that binds to the promoter/
operator region (P–O). When the repressor is
bound to the P–O region, RNA polymerase cannot
bind to the promoter region. Transcription
is blocked (1), and the three gene products are
not formed.The lac operon is activated when a
!-galactoside molecule binds to one of the subunits
of the repressor (2) and inactivates it. RNA
polymerase can then bind to the promoter region
and transcription can begin.

Gene-regulating nucleotide sequence of the lac operator

The activity of a gene is mediated by gene regulatory
proteins that bind to DNA at specific sites.
The repressor of the lactose operon (lac repressor)
was the first such protein to be isolated (in
1966 by Gilbert and Müller-Hill). The repressor
is a tetramer of identical 37-kDa subunits. Each
has a binding site for the inducer. In the absence
of the inducer, the repressor binds very tightly
to the DNA in the operator region. The recognition
sequence of the repressor is a short
sequence of 28 nucleotide base pairs that are related
by an axis of symmetry (shown as colored
boxes). Symmetry matching is an important
principle of protein–DNA interaction at a
genetic switch. Gene-regulatory proteins can be
dinstinguished by the specificity of the DNA
sequences they recognize

Control of Gene Expression in Bacteria by Repression

If a gene is usually expressed (is active), it is said
to be constitutive. Gene expression in bacteria
can vary considerably depending on the presence
or absence of certain substances in the
nutrient medium. An important mechanism for
controlling transcription is a signal (termination
signal) that can terminate transcription or
translation. It lies between the promoter and
the beginning of the first structural gene and is
called the attenuator (attenuation of translation).

Regulation of synthesis of the amino acid tryptophan in E. coli

Tryptophan is an essential amino acid in
eukaryotic organisms. Bacteria can synthesize
tryptophan, but will do so only when it is not
present in the nutrient medium (2). If tryptophan
is added to the medium, enzyme activity
for tryptophan biosynthesis decreases
within about 10 minutes

Biosynthesis of tryptophan by means of five enzymes and five genes

Tryptophan is synthesized from chorismate via
four intermediates; this occurs in five steps,
regulated by five enzymes. The enzymes are
coded for by five genes (trpA–E) (CdRP = carboxyphenylamino-
deoxyribulose-phosphate).

Tryptophan operon in E. coli

The tryptophan operon in E. coli consists of
these five genes and their regulating sequences.
The latter include promoter and operator, a
leader sequence, and attenuator sequences.
Translation of the five structural genes results
from a continuous trp operon mRNA. In this,
leader sequences coded for by L-sequence
genes are connected in series. The attenuator
sequences are part of the L-sequences. When
tryptophan is present in the medium, translation
of trp leader RNA is discontinued in the region
of an attenuator sequence, before reaching
the first structural gene.

The role of the attenuator

The weakening (attenuation) of the expression
of the tryptophan operator is controlled by a
sequence of about 100–140 base pairs (in the 3!
direction) from the starting point of transcription
(tryptophan mRNA leader). In the presence
of tryptophan, the trp mRNA leader is interrupted
in the region of an attenuator sequence
(1), and translation does not take place. In the
absence of tryptophan, translation is continued.
The trp leader peptide contains two tryptophan
residues (2). When tryptophan is deficient,
translation is delayed and a stop signal
weakened.

Attenuation of the trp operon

Attenuation of the trp operon
In E. coli, attenuation is often mediated by a
tight relation of transcription and translation.
The trp mRNA leader region can exist in two alternative
base-pair conformations. One allows
transcription, the other not. When tryptophan
ist present (1), ribosomes can synthesize the
complete leader peptide. The ribosome closely
follows the RNA polymerase transcribing the
DNA template (not shown). The ribosome has
passed region 1 and prevents complementary
regions 2 and 3 from forming a hairpin by base
pairing. Instead, part of complementary region
3 and region 4 form a stem and a loop, which
favors termination of transcription. When tryptophan
is deficient (2), the ribosome stalls at the
two UGG trp codons owing to deficiency of
tryptophanyl-tRNA. This alters the conformation
of the mRNA so that regions 2 and 3 pair.
The stem structure favoring termination is not
formed, region 4 remains single-stranded, and
transcription continues.

Control of Transcription

Transcription is controlled at promoters and
other DNA sequences (enhancers) outside of
the actual coding region. Transcription control
in prokaryotes and eukaryotes differ in some respects
and correspond in others. An important
region for controlling gene expression, the promoter
region, lies upstream (5! direction) of the
coding sequence.

Promoter region

In prokaryotes, two important areas in the promoter
region are 35 and 10 nucleotide base
pairs upstream of (in the 5! direction) the
starting point of transcription. Mutations in the
region of the regulative sequences (promoter
region) in certain regions are extremely sensitive
to base substitution (mutation), an indication
of their importance.

Assembly of general transcription factors to initiate transcription

The activation of polymerase II (Pol II) to transcribemost
eukaryotic genes (1) requires an initiation
complex assembled at the promoter. It
consists of general transcription factors (TF)
that associate in an ordered sequence. In the
first step (2), TFIID (transcription factor D for
polymerase II) binds to the TATA region. The
TATA box is recognized by a small, 30-kDa
TATA-binding protein (TBP), which is part of
one of the many subunits of TFIID (the bending
of the DNA by TBP is not shown here).
Following this, TFIIB can bind to the complex
(3). Subsequently, other transcription factors
(TFIIH, followed by TFIIE) and Pol II escorted by
TFIIF join the complex and assure that Pol II is
attached to the promoter (4). The binding of
TFIIE extends the polymerase binding sites
further downstream in the 3! direction. Pol II is
then released from the complex and transcription
can begin. A key step is phosphorylation of
Pol II by a subunit of TFIIH,which is a protein kinase.
Other activities of TFIIH involve a helicase
and an ATPase. The site of phosphorylation is a
polypeptide tail, composed in mammals of 52
repeats of the amino acid sequence YSPTSPS in
which the serine (S) and threonine (T) side
chains are phosphorylated.

RNA polymerase promoters

Eukaryotic cells contain three RNA polymerases
(Pol I, Pol II, and Pol III). Pol I is located in the
nucleolus, synthesizes ribosomal RNA, and accounts
for about 50–70% of the relative activity.
Pol II and Pol III are located in the nucleoplasm
(the part of the nucleus excluding the nucleolus).
Pol II represents 20–40% of cellular activity.
It is responsible for the synthesis of heterogeneous
nuclear RNA (hnRNA), the precursor of
mRNA. Pol III, responsible for the synthesis of
tRNAs and other small RNAs, contributes only a
minor activity of about 10%. Each of the large
eukaryotic RNA polymerases (500 kDa or more)
is more complex, with 8–14 subunits, than the
single prokaryotic RNA polymerase.

RNA polymerase

Each RNA
polymerase uses a different type of promoter.
RNA polymerase II cannot initiate transcription
without a complex of general transcription factors
(see B) that bind to a single upstream promoter
(1). RNA polymerase I (2) has a bipartite
promoter, one 170 to 180 bp upstream (5! direction)
and another from about 45 bp upstream to
20 bp downstream (3! direction). The latter is
called the core promoter. Pol I requires two ancillary
factors, UBF1 and SL1. SL1 consists of four
proteins including a TBP (see B) that cannot
bind directly to the promoter. It binds to UBF1,
after which Pol I can bind to the core promoter
to initiate transcription (2). RNA polymerase III
uses either upstream promoters or two internal
promoters downstream of the transcription
start site (3). Three transcription factors are required
with internal promoters, TFIIIA (a zinc
finger protein, see p. 218), TFIIIB (a TBP and two
other proteins), and TFIIIC (a large, more than
500 kDa protein).