Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-03-30
2003-09-02
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
Reexamination Certificate
active
06613531
ABSTRACT:
BACKGROUND OF THE INVENTION
The RNA polymerase of
Escherichia coli
is a large, multisubunit enzyme existing in two forms. The core enzyme, consisting of subunits &bgr; and &bgr;′ and an a subunit dimer, carries out processive transcription elongation followed by termination (Helmann et al., 1988). When one of a variety of sigma (&sgr;) factors is added to core, the holoenzyme is formed (Burgess et al., 1969). The &sgr; subunit confers promoter-specific DNA binding and transcription initiation capabilities to the enzyme (Helmann et al., 1988; Burgess et al., 1969; Gross et al., 1996; Gross et al., 1992). &sgr;
70
of
E. coli
was the first a factor to be described and characterized (Burgess et al., 1969). Since then, numerous a factors have been discovered throughout the Eubacterial kingdom, including six alternative a factors in
E. coli.
Each &sgr; subunit directs its cognate holoenzyme to start transcription from only those promoters containing DNA sequences specifically recognized by the &sgr; factor. Thus, generally, each a directs transcription initiation from a specific set of promoters to transcribe genes with related functions. This control of transcription is mediated partially through the competition of the individual &sgr; factors for the core enzyme and is a major part of global gene regulation in bacteria (Zhou et al., 1992).
As the number of identified &sgr; factors increased, it became apparent that they shared several regions of amino acid sequence similarity (Helmann et al., 1988; Gribskov et al., 1986; Lonetto et al., 1992), and the function of the conserved regions is of continuing interest (Waldburger et al., 1994; Dombrowski et al., 1993; Siegele et al., 1989; Gardella et al., 1989; Lesley et al., 1989). Deletion analysis of &sgr;
70
identified a segment of the protein that overlaps conserved region 2.1 (residues 361-390) as being necessary and sufficient for core binding (Lesley et al., 1989). A mutation in a homologous region of
Bacillus subtilis &sgr;
E
has also been shown to affect core binding (Shuler et al., 1995). However, recent findings of core binding mutations in other conserved and nonconserved regions of &sgr;
32
have led to the idea of multiple binding sites for the &sgr; subunit on the core enzyme (Joo et al., 1997; Zhou et al., 1992; Joo et al., 1998; Sharp et al., 1999).
The &bgr; and &bgr;′ subunits each contain regions that have high sequence homology with the two largest subunits of eukaryal polymerases (Allison et al., 1985; Sweetser et al., 1987; Jokerst et al., 1989). Some of these conserved regions may act as interaction domains. An interaction domain is the minimal region of a protein that can independently fold to form the secondary and tertiary structure required to interact with another protein, DNA, RNA, or ligand. Interaction domains are larger than the actual binding site which is formed by the amino acids in direct contact with the binding partner. Severinov et al. (Severinov et al., 1992, 1995 and 1996) demonstrated the domain-like properties of &bgr; and &bgr;′ by reconstitution of functional RNA polymerase from fragmented &bgr; and &bgr;′ subunits. Thus, the properties of the polymerase do not require the entire intact length of the subunit but rather can be generated with smaller domain modules.
There have been two observations that have identified deletions in the &bgr; or &bgr;′ subunits that produce subunits still capable of forming core enzyme structures but not the holoenzyme. First, a &bgr; subunit truncation, missing approximately 200 amino acids of the C terminus, was shown by glycerol gradient centrifugation to migrate with the other core subunits but was never seen in the &sgr;-containing fractions (Glass et al., 1986). Second, when immunoprecipitation assays were performed using reconstituted RNA polymerase containing &bgr;′ deletion mutants missing amino acids 201-477, the core subunits were recovered in the same fraction but lacked &sgr; (Luo et al., 1996). However, it was unclear whether the &bgr;′ deletion was non-specific, e.g., prevented correct formation of the interaction domain.
The idea that &sgr; binding is affected by perturbations of the C terminus of &bgr; and the N terminus of &bgr;′ is consistent with experiments showing that these two subunit termini are physically close together and can be fused through a flexible linker and still form a functional enzyme (Severinov et al., 1997). Recent protein-protein footprinting data have identified a similar region on &bgr;′ and two new sites on &bgr; for possible interactions with the &sgr;
70
subunit (Owens et al., 1998). While Owens et al. showed that residues 228-461 of &bgr;′ are physically close to &sgr;, the authors did not conclude that there is a direct interaction between &bgr;′ and &sgr;.
Burgess et al. (1998) report that residues 260 to 309 of &bgr;′ bind to a based on the use of in vitro far-Western and co-immobilization assays. However, in vitro cell-free binding results do not evidence that the region involved in binding in vitro is involved in binding in vivo. For example, it is possible that this region of &bgr;′ is buried in the native structure, e.g., a hydrophobic region, and so would not play a role in vivo binding. Structural analysis programs indicate that &bgr;′
260-309
has two &agr; helices joined by a random coil, and that these two helices are amphipathic and have the potential for coiled coil formation, based on a heptad repeat motif (Chao et al., 1998; Cohen et al., 1986; Lupas et al., 1991). In particular certain positions known as a and d in the coiled coil motif are hydrophobic and so may be buried in native &bgr;′
Thus, what is needed is the identification of a region in the subunits of core RNA polymerase that interacts with &sgr; in vivo. What is also needed is a method to identify specific inhibitors of the binding of &sgr; to core RNA polymerase.
SUMMARY OF THE INVENTION
The invention provides an isolated and purified &bgr;′ subunit of RNA polymerase or a portion (i.e., fragment) thereof which specifically binds to &sgr; in vivo. Preferably, the portion comprises at least 39, more preferably at least 44, and even more preferably at least 49, residues of the &bgr;′ subunit, although smaller fragments which specifically bind to &sgr; in vivo are also envisioned. Also preferably, the isolated and purified portion of the &bgr;′ subunit comprises residues 270 to 309, and even more preferably residues 260 to 309. As described hereinbelow, a region on the &bgr;′ subunit of RNA polymerase was identified that interacts directly with &sgr; (the interaction domain). The in vitro interaction domain of the &bgr;′ subunit with &sgr; was identified by far-Western blot analysis, which is a general method for mapping a domain on one protein that is necessary for binding another protein, and a co-immobilization assay. As used herein, an “interaction domain” refers to the minimal region of a protein that can independently fold to form the secondary and tertiary structures required to interact with another protein, DNA, RNA or ligand. The &sgr; binding region of &bgr;′ was found to interact with various a factors, including &sgr;
70
and several other
E. coli
&sgr;'s, T4 phage &sgr; gp 55, and &sgr;
A
from
Bacillus subtilis.
As also described hereinbelow, proteins were prepared which had single point mutations in the predicted coiled coils located within residues 260-309 of &bgr;′. Several of the mutants were defective for binding &sgr;
70
in vitro. Of these mutants, three (R275Q, E295K, and A302D which are change-of-charge mutants at the e and g residues of the &bgr;′
260-309
predicted coiled coil) were completely defective for growth in an in vivo assay where the mutant &bgr;′ is the sole source of &bgr;′ subunit. All of the mutants were able to assemble into the core enzyme, however, R275Q, E295K, and A302D were defective for E&sgr;
70
-holoenzyme formation. Severa
Anthony Larry C.
Arthur Terrance M.
Bergendahl Veit
Burgess Richard R.
Pietz Bradley C.
Horlick Kenneth R.
Schwegman Lundberg Woessner & Kluth P.A.
Wisconsin Alumni Research Foundation
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