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