Mutant of RNA polymerases with increased stability

Details Mutant of RNA polymerases with increased stability Mutant of RNA polymerases with increased stability

2001-06-05

2003-02-25

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S320100, C435S325000, C536S023100, C536S023200

Reexamination Certificate

active

06524828

ABSTRACT:

STATEMENT OF FEDERAL SUPPORT
Not Applicable
FIELD OF THE INVENTION
The present application relates to mutated RNA polymerases from bacteriophages that have increased stability, for example under high temperature conditions. One example of bacteriophage encoded RNA polymerase is the T7 RNA polymerase. T7 is a bacteriophage capable of infecting
E. coli
cells. Examples of other
E. coli
infecting T7-like bacteriophages are T3, &phgr;I, &phgr;II, W31, H, Y, A.1, croC21, C22 and C23. An example of a
Salmonella typhimurium
infecting bacteriophage is SP6.
BACKGROUND OF THE INVENTION
The RNA polymerases of bacteriophages have high selectivity for their own promoter sequence. The T7 RNA polymerase will bind the T7 RNA polymerase promoter sequence but not one of the other bacteriophage promoter sequences. The high promoter specificity ensures that the bacteriophage transcription reaction is only directed to its own genome and not the host genome. The entire nucleotide sequence of the T7 bacteriophage is known and the phage RNA polymerase is encoded by T7 gene 1. Other RNA polymerases that resemble the T7 RNA polymeraselare the RNA polymerases of bacteriophages SP6 and T3. The T3 RNAP shows about 80% homology with the T7 RNAP.
The T7 gene 1 has been cloned and expressed in bacteria allowing the production of large quantities of the enzyme (Studier et al., U.S. Pat. No. 4,952,496). The T7 98,6 Kda. T7 RNA polymerase does not require any auxiliary factors for accurate transcription. The enzyme alone is capable of recognising it's promoters, initiating transcription, elongating the RNA transcript and terminating transcription. T7 RNA polymerase is very efficient in transcribing DNA from its own promoters and elongates RNA five times faster compared to
E. coli
RNA polymerase. Their selectivity, activity and ability to produce complete transcripts make the polymerases from bacteriophages very useful for a variety of purposes.
The present invention is concerned with the RNA polymerases of T7-like bacteriophages that have been mutated.
Some specific mutants of T7-like bacteriophage RNA polymerases' have been described. For example, in WO91/05866 an alternative expression system is described. The system is an attempt to use the bacteriophage T7 promoters to direct the transcription of a cloned gene in bacteria. The system uses a truncated T7 RNA polymerase, the gene of which is mutated by deleting a nucleotide (one or more bases corresponding to base 3809 and 3877 of a wild type T7 polymerase gene). This deletion results in a frame shift and consequently a new translation stop codon is created. In U.S. Pat. No. 5,385,834, a mutant T7 RNAP is also described. The mutant described in U.S. Pat. No. 5,385,834 is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. This mutant exhibit altered promoter recognition, and thus the mutant is able to initiate transcription from T7 promoter point mutations that are normally inactive.
Ikeda et al. (Ikeda, R. A. et al. Biochemistry, 31:9073-9080, 1992 and Ikeda, R. A. et al., Nucl. Acid. Res., 20: 2517-2524,1992) have described two compatible plasmids that can be used for screening the activity of mutated T7 RNAP, gene- or promoter sequences. The first plasmid carries the T7 gene 1 (the gene encoding the T7 RNA polymerase) ligated to an
E. coli
tac promoter., while the second plasmid carries the gene encoding CAT (chloramphenicol acetyl transferase) ligated to the T7 promoter.
E. coli
cells carrying these two plasmids are CAM (chloramphe Inicol) resistant if the T7 polymerase interacts with the T7 promoter and transcribes the CAT gene from the second plasmid. If either the T7 promoter or the T7 RNA polymerase is inactive, the CAT gene will not be transcribed and thus the
E. coli
cells will be cam sensitive. Ikeda et al. used the plasmids to investigate the effects of certain mutations on the activity of T7 RNA polymerase promoters. With a plasmid system like the one described by Ikeda et al., where the T7 RNA polymerase gene 1 is on one plasmid under the control of a suitable promoter, and the T7 RNA polymerase promoter is on a second plasmid controlling a resistance gene like CAT, mutant T7 RNA! polymerases itself can be screened for their activity as well.
In vitro transcription with the aid of bacteriophage encoded RNA p6lymerases (e.g. T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymeras has become a widely applied tool in molecular biology. Next to the in vitro transcription on its own, as a tool to make fast amounts of RNA bacteriophage, RNA polymerases are part of nucleic acid amplification methods. Such methods are for instance NASBA, 3SR and TMA. In vitro transcription has also. been described in combination with PCR as an extra linear amplification step post PCR amplification. l
For all of the above applications it would be advantageous if the reaction temperature could be elevated so that the kinetics of the transcription reaction becomes better and more importantly that isothermal amplification methods (NASBA, 3SR and TMA) can be performed at higher temperatures. This higher incubation temperature of the isothermal amplification reaction will enable the amplification of structured RNA's more efficiently. Applications where this is important are amplification of long RNA sequences (>500 nucleotides) and multiplex reactions (i.e. the amplification of multiple RNA sequences in one reaction mixture).
BRIEF SUMMARY OF THE INVENTION
The present invention relates to mutants of T7 like bacteriophage derived RNA polymerases that have an increased stability.
Analysis of randomly mutated T7 RNA polymerase mutants revealed a number of possible mutations that have a stabilizing effect on the T7 RNA polymerase protein and enable enzymatic activity at higher temperatures than normal (normal is 37° C.-41° C.). The randomly mutated T7 RNA polymerase sequences were analyzed by screening the sequences in a two plasmid system as described by Ikeda et al (1992) in
Bacillus stearothermophilus.
The
Bacillus stearothermophilus
cells were grown at elevated temperatures (45°-50° C.) and CAM resistance could only be obtained if a mutated T7 sequence would encode a more stable T7 RNA polymerase capable of polymerase activity at these temperatures. In the
Bacillus stearothermophilus
system one plasmid contains an antibiotic resistance gene (CAT) under control of the T7 promoter and the other plasmid contains a mutant library of the T7 RNA polymerase under control of a Bacillus promoter. In those cases where the mutation allows the T7 RNA polymerase to be functional at the elevated temperature the
Bacillus stearothermophilus
will have become CAM resistant. Using the above described system 43 clones of the T7 RNA polymerase gene were found. Of this collection, 12 clones were analyzed in more detail, i.e. the nucleotide sequence of the encoding gene determined. The collection of 11 analyzed clones consisted of both mutations leading to amino acid changes and silent mutations (see table 1). The mutations leading to amino acid changes were investigated further.
TABLE 1
Summary of T7 RNA polymerase mutations leading to increased
stability in a
Bacillus stearothermophilus
two plasmid
screening system.
Nucleotide
Nucleotide
Amino acid
Amino acid
Mutant name
mutation
numbers
1
mutation
numbers
2
S1
3
?
?
3-8
 A→T,
65
N→I
 22
A→T
884
A→V
295
4-5
A→T
1748
Q→L
583
4-7
A→T
64
N→Y
 22
A→G
80
H→R
 27
A→C
136
M→L
 46
T→C
521
V→A
174
A→T
999
K→N
333
7-7
T→C
1897
S→P
633
T→C
2499
Silent
—
3-13
T→A
387
silent
—
3-19
T→C
2202
silent
—
4-3
T→C
2520
silent
—
4-10
T→C
2520
silent
—
4-6
C→T
453
silent
—
7-1
T→C
225
silent
—
1
Nucleotide numbering is according to Dunn, J. J. and Studier, F. W. [(1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements J. Mol. Biol. 166 (4), 477-535] with numbe

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