Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-03-01
2003-08-26
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Vector, per se
C530S333000
Reexamination Certificate
active
06610533
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the control of bacterial gene expression, especially the regulation of bacterial gene expression under conditions of physiologic stress. More specifically, the invention relates to the regulation of bacterial gene expression under conditions of physiologic stress that induce the cold shock response of a bacterium.
2. Description of the Related Art
The regulation of bacterial gene expression occurs at many levels, including transcriptional control, or control of the synthesis of mRNA from a given gene; translational control, or the regulation of the efficiency by which the mRNA is translated into polypeptide sequence by the ribosome; and mRNA stability, or the efficiency at which a given mRNA population within the cell is degraded and rendered inactive. The control of bacterial gene expression under conditions of physiologic stress that elicit the cold shock response of the bacterium involve regulation at all three of the levels described above.
The response of bacteria to physiologic stress involve the tightly controlled expression of a small number of genes that function to allow the cell to adapt to and multiply under stress conditions. For example, when bacterial cells are exposed to temperatures above the normal physiologic temperature for that organism, a set of genes, designated the heat shock genes, are expressed. This response to elevated temperatures is well known and described in the prior art. Conversely, when bacterial cells are exposed to lower than physiologic temperatures, a different set of genes, designated as cold shock (cs) genes, are expressed. Expression of the cs genes allow the cell to first adapt to the physiologic stress, and subsequently grow under conditions of physiologic stress. This invention relates to the specific processes that regulate the expression of cs genes.
When a culture of
Escherichia coli
is shifted from 37° C. to 15 or 10° C., a number of proteins, called cold-shock proteins, are transiently induced during its growth lag period (Jones et al., 1987; for review, see Thieringer et al., 1998; Yamanaka et al., 1998). cspA, consisting of 70 amino acid residues, has been identified as a major cold-shock protein (Goldstein et al., 1990) and its three-dimensional structure has been determined by both X-ray crystallography (Schindelin et al., 1994) and nuclear magnetic resonance spectroscopy (Newkirk et al., 1994; Feng et al., 1998) to consist of a five-antiparallel &bgr;-stranded structure. cspA can bind to single-stranded DNA and RNA without high sequence specificity and has been proposed to function as an RNA chaperone at low temperature (Jiang et al., 1997).
To date, more than 50 proteins homologous to cspA have been identified in a large varieties of prokaryotes. Moreover, a region called cold-shock domain of eukaryotic Y-box protein family, such as human YB-1 and Xenopus FRGY-2, shares more than 40% identity with
E. coli
cspA (for review, see Wolffe et al., 1992), indicating that the cold-shock domain is well conserved throughout evolution. In
E. coli
, nine genes encoding cspA-like proteins, cspA to cspI, have been identified (for review, see Yamanaka et al., 1998). Among them, cspA, cspB and cspG are cold-shock inducible (Goldstein et al., 1990; Lee et al., 1994; Nakashima et al., 1996) and interestingly, cspD is induced during stationary phase and upon nutrition starvation (Yamanaka and Inouye, 1997). It was proposed that the large cspA family of
E. coli
may have a function to respond to different environmental stresses (for review, see Yamanaka et al., 1998).
cspA expression is transiently induced upon cold shock during the growth lag period called acclimation period. This period is considered to be required for cells to adapt to a new environmental condition. Indeed, during the acclimation period proteins involved in translation such as csdA (Jones et al., 1996), RbfA (Jones and Inouye, 1996) and cspA (Goldstein et al., 1990) are specifically produced, which are considered to play important roles in enhancing translation efficiency for non-cold-shock proteins at low temperature (for review, see Yamanaka et al., 1998). Among these cold-shock proteins, cspA has been quite extensively investigated for the mechanism of its cold-shock induction (for review, see Yamanaka et al., 1998). The cspA promoter is highly active at 37° C., even if cspA is hardly detected at this temperature (Fang et al., 1997; Mitta et al., 1997). Even if the cspA promoter was replaced with the lpp promoter, a constitutive promoter for a major outer membrane protein, cspA expression is still cold-shock inducible (Fang et al., 1997), indicating that the cspA induction at low temperature occurs mainly at levels of mRNA stability and its translation. The cspA promoter, however, contains an AT-rich upstream element (UP element) (Ross et al., 1993) immediately upstream of the −35 region (Fang et al., 1997; Goldenberg et al., 1997; Mitta et al., 1997), which is considered to play an important role in efficient transcription initiation at low temperature. It has been demonstrated that the cspA mRNA becomes extremely stable upon cold shock, indicating that the mRNA stability plays a crucial role in cold-shock induction of cspA (Brandi et al., 1996; Goldenberg et al., 1996; Fang et al., 1997).
An important and unique feature of the cspA mRNA is its unusually long 5′-untranslated region (5′-UTR) consisting of 159 bases (Tanabe et al., 1992). This feature is also shared with other Class I cold-shock genes, which are dramatically induced after temperature downshift (for review, see Thieringer et al., 1998), such as cspB (Etchegaray et al., 1996) and cspG (Nakashima et al., 1996). The 5′-UTR is considered to play a crucial role in the cold-shock induction of cspA (Brandi et at., 1996; Jiang et at., 1996; Goldenberg et al., 1996; Bae et al., 1997; Fang et al., 1997; Goldenberg et al., 1997; Mitta et al., 1997).
Furthermore, it was recently shown that the 14-base downstream box (DB) located 12 bases downstream of the rt codon of the cspA mRNA, which is partially complementary to a region called anti-downstream box of 16S rRNA (Sprengart et al., 1996), plays an important role in efficient translation at low temperature (Mitta et al., 1997). This region of the RNA sequence designated as the downstream box (DB) is complementary to bases 1469-1483 within the
E. coli
16S rRNA (anti-DB sequence). It is speculated that formation of a duplex between the DB and anti-DB of 16S rRNA is responsible for translational enhancement (Sprengart et al., 1996). The DB sequence has also been implicated in the translation of the mRNA, a mRNA that lacks any untranslated region and the SD sequence (Shean and Gottesman, 1992; Powers et al., 1988). Interestingly &lgr;c1 translation was enhanced at 42° C. in a temperature sensitive strain in which the amount of ribosomal protein S2 decreased at 42° C. It was proposed that the anti-DB sequence in S2 deficient ribosomes indirectly becomes more accessible to DB, resulting in enhancement of translation initiation of the &lgr;c1 mRNA. However, the role of the DB in &lgr;c1 translation initiation was disputed by Resch and coworkers (Resch et al., 1996). These authors constructed lacZ translational fusions with the &lgr;c1 gene to test the DB function. Since a deletion of 6 bases encompassing a portion of the DB sequence did not reduce the formation of the translation initiation complex, they disputed the existence of DB. Despite these elusive roles of DB (Sprengart and Porter, 1997) we have shown that the presence of a DB sequence in cold-shock mRNAs plays an important role in translation efficiency, and proposed that the DB is involved in the formation of a stable initiation complex at low temperature before the induction of cold-ribosomal factors (Mitta et al., 1997). Thus, cspA expression is regulated in a complex manner at levels of transcription, mRNA stability and translation.
REFERENCES:
Blattner et al. 1977; Science 277:1453-1474.*
Wang et
Inouye Masayori
Wang Nan
Yamanaka Kunitoshi
Carlson Karen Cochrane
Piper Rudnick LLP
University of Medicine and Dentistry of New Jersey
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