Biomedical Engineering Reference
In-Depth Information
this consensus sequence consists of the
−
35 region
U residues at the 3
end of the mRNA. The factor-
dependent transcription terminators have very little
sequence in common with each other. Rather,
termination involves interaction with one of the
three known
E. coli
termination factors, Rho (
′
(5
′
-TTGACA-) and the
−
10 region, or Pribnow box
(5
-TATAAT). RNA polymerase must bind to both
sequences to initiate transcription. The strength of a
promoter, i.e. how many RNA copies are synthes-
ized per unit time per enzyme molecule, depends on
how close its sequence is to the consensus. While
the
′
ρ
),
Tau (
) and NusA. Most expression vectors incor-
porate a factor-independent termination sequence
downstream from the site of insertion of the cloned
gene.
τ
10 regions are the sites of nearly all
mutations affecting promoter strength, other bases
flanking these regions can affect promoter activity
(Hawley & McClure 1983, Dueschle
et al.
1986,
Keilty & Rosenberg 1987). The distance between the
−
−
35 and
−
Vectors for making RNA probes
10 regions is also important. In all cases
examined, the promoter was weaker when the
spacing was increased or decreased from 17 bp.
Upstream (UP) elements located 5
35 and
−
Although single-stranded DNA can be used as a
sequence probe in hybridization experiments, RNA
probes are preferred. The reasons for this are that the
rate of hybridization and the stability are far greater
for RNA-DNA hybrids compared with DNA-DNA
hybrids. To make an RNA probe, the relevant gene
sequence is cloned in a plasmid vector such that it
is under the control of a phage promoter. After
purification, the plasmid is linearized with a suitable
restriction enzyme and then incubated with the
phage RNA polymerase and the four ribonucleoside
triphosphates (Fig. 5.8). No transcription termin-
ator is required because the RNA polymerase will fall
off the end of the linearized plasmid.
There are three reasons for using a phage pro-
moter. First, such promoters are very strong, enabling
large amounts of RNA to be made
in vitro
. Secondly,
the phage promoter is not recognized by the
E. coli
RNA polymerase and so no transcription will occur
inside the cell. This minimizes any selection of vari-
ant inserts. Thirdly, the RNA polymerases encoded
by phages such as SP6, T7 and T3 are much simpler
molecules to handle than the
E. coli
enzyme, since
the active enzyme is a single polypeptide.
If it is planned to probe RNA or single-stranded
DNA sequences, then it is essential to prepare RNA
probes corresponding to both strands of the insert.
One way of doing this is to have two different clones
corresponding to the two orientations of the insert.
An alternative method is to use a cloning vector in
which the insert is placed between two different,
opposing phage promoters (e.g. T7/T3 or T7/SP6)
that flank a multiple cloning sequence (see Fig. 5.5).
Since each of the two promoters is recognized by a
different RNA polymerase, the direction of transcrip-
tion is determined by which polymerase is used.
35 hex-
amer in certain bacterial promoters are A
′
of the
−
T-rich
sequences that increase transcription by interacting
with the
+
subunit of RNA polymerase. Gourse
et al.
(1998) have identified UP sequences conferring
increased activity to the
rrn
core promoter. The best
UP sequence was portable and increased heterolo-
gous protein expression from the
lac
promoter by a
factor of 100.
Once RNA polymerase has initiated transcription
at a promoter, it will polymerize ribonucleotides
until it encounters a transcription-termination
site in the DNA. Bacterial DNA has two types of
transcription-termination site: factor-independent
and factor-dependent. As their names imply, these
types are distinguished by whether they work with
just RNA polymerase and DNA alone or need other
factors before they can terminate transcription.
The factor-independent transcription terminators
are easy to recognize because they have similar
sequences: an inverted repeat followed by a string of
A residues (Fig. 5.7). Transcription is terminated
in the string of A residues, resulting in a string of
α
GC - rich inverted repeat
5' -G
C
T
A
C
G
A
T
A
T
A
T
A
T
G
C
C
G
C
G
T
A
C
G
C
G
G
C
G
C
T
A
C
G
G
C
G
C
A
T
G
C
G
C
C
G
T
A
T
A
T
A
T
A
G
C
A
T
C
G
T
A-
5'
Run of
A residues
Fig. 5.7
Structure of a factor-independent transcriptional
terminator.
