section 25.4
Prokaryotic Transcription
567
C G T A T A A IG T G T G G
G G T A C G A T G T A C C A C
A G T A A G A I A C A A A T C
GT G A T A A J G G T T G C
C T T A T A A I G G T T A C
C G T A T G T I G T G T GG
G C T A T G G IT A T T T C
G T T T T C A I G C C T C C
A G G A T A C IT AC A G C O
T G T A T A A I A G A T T
C
G G C A T G A T A G C G C C C
G C T T T A A T G C G G T A
FIGURE 25-4
Segments of the coding strand of conserved regions from various genes
showing the common sequence of six bases. The start point for mRNA
synthesis is indicated by the heavy letters. The “conserved T” is underlined.
a
promoter
—a specific nucleotide sequence, which for
different RNA classes range in length from 20 to 200 nu-
cleotides. In bacteria, a promoter is divided into subregions
called the -35 sequence (8-10 bases long) and the -10 or
Pribnow box
( 6
bases). The bases in the -35 sequence are
quite variable but the Pribnow boxes of all promoters are
similar (Figure 25-4). Both the -35 sequence and the Prib-
now box interact with RNA polymerase to initiate RNA
synthesis.
Following RNA polymerase binding to a promoter, a
conformational change occurs such that a segment of the
DNA is unwound and RNA polymerase is positioned at
the polymerization start site. Transcription begins as soon
as the RNA polymerase-promoter complex forms and an
appropriate nucleotide binds to the enzyme. RNA poly-
merase contains two nucleotide binding sites called the
initiation site
and the
elongation site.
The initiation site
binds only purine triphosphates (ATP and GTP), and one
of these (usually ATP) becomes the first nucleotide in the
RNA chain. Thus, the first DNA base that is copied from
the DNA is usually thymine. The initiating nucleoside
triphosphate binds to the enzyme and forms a hydrogen
bond with the complementary DNA base. The elongation
site is then filled with a nucleoside triphosphate that is se-
lected strictly by its ability to form a hydrogen bond with
the next base in the DNA strand. The two nucleotides are
then joined together, the first base is released from the ini-
tiation site, and initiation is complete. The dinucleotide
remains hydrogen-bonded to the DNA. The elongation
phase begins when the polymerase releases the base and
then moves along the DNA chain.
The drug
rifampin
binds to bacterial RNA polymerases
and is a useful experimental inhibitor of initiation of tran-
scription. It binds to the
ft
subunit of RNA polymerase,
blocking the transition from the chain initiation phase to
the elongation phase; it is an inhibitor of chain initiation
but not of elongation.
Actinomycin D
also inhibits initi-
ation but does so by binding to DNA. These drugs have
limited clinical use because of their toxicity.
FIGURE 25-5
Base sequence of (a) the DNA of the
E. coli
trp operon at which
transcription termination occurs and of (b) the 3' terminus of the mRNA
molecule. The inverted-repeat sequence is indicated by reversed arrows.
The mRNA molecule is folded to form a stem-and-loop structure.
After several nucleotides have been added to the grow-
ing chain, RNA polymerase holoenzyme changes its
structure and loses the
o
subunit. Thus, most elongation
is carried out by the core enzyme, which moves along the
DNA, binding a nucleoside triphosphate that can pair with
the next DNA base and opening the DNA helix as it moves.
The open region extends over about 30 base pairs. Chain
elongation does not occur at a constant rate but slows down
or stops at various points along the DNA molecule and, in
some cases, may have a regulatory function.
Termination of RNA synthesis occurs at specific base
sequences within the DNA molecule. Many prokaryotic
termination sequences have been determined and most
have the following three characteristics (Figure 25-5):
1. An inverted-repeat base sequence containing a central
nonrepeating segment; the sequence in one DNA
strand would read ABCDHF-XYZ-F'E'D'C'B'A', in
which A and
A',
B and B', and so on, are
complementary bases. The RNA transcribed from this
segment is capable of intrastrand base pairing,
forming a stem and loop.
2. A sequence having a high G + C content.
3. A sequence of AT pairs in DNA (which may begin in the
stem) that results in a sequence of
6 - 8
U’s in the RNA.
Termination of transcription includes the following
steps:
