DEAH-box proteins

Details DEAH-box proteins DEAH-box proteins

1999-06-23

2001-06-26

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C435S194000, C435S195000, C435S196000, C536S023200, C530S350000

Reexamination Certificate

active

06251645

ABSTRACT:

BACKGROUND OF THE INVENTION
The modulation of RNA structure is an essential regulatory process in many cellular events, such as, for example, pre-mRNA splicing, assembly of spliceosomes, assembly of ribosomes, protein translation, which can be summarized under the generic term “regulation of gene expression at the RNA level”. The so-called “DEAD box” protein family of putative RNA helicases, named after the characteristic amino acid motif Asp-Glu-Ala-Asp (in the single-letter code DEAD), in this context plays a key part (in particular for the modulation of the secondary and tertiary structure of MRNA). DEAD box proteins are also involved in processing of DNA. The members of this family and some subfamilies have differences in their specific function and cellular localization. However, in addition to characteristic sequence homologies certain members also show similar biochemical properties (F. V. Fuller-Pace, Trends in Cell Biology, Vol 4, 1994, 271-274). The characteristic protein sequences of the DEAD proteins are highly conserved in evolution (S. R. Schmid and P. Lindner, Molecular and Cellular Biology, Vol 11, 1991, 3463-3471). Members of this protein family are found in various viruses, bacteria, yeasts, insects, molluscs and lower vertebrates up to mammals and are responsible for a large number of cellular functions. The fact that even relatively simple organisms such as, for example, the yeast
Saccharomyces cerevisiae
express numerous proteins of the DEAD box protein family and their subfamilies, suggests that each of these proteins contributes to the specific interaction with certain RNAs or RNA families (I. Iost and M. Dreyfus, Nature Vol 372, 1994, 193-196). It has been shown that translation factors, such as elF-4A and the proteins involved in the pre-MRNA splicing process, recognize specific RNA target sequences or structures. Nevertheless, to date there is little information about the structure and the synthesis of characteristic RNA sequences which require the DEAD proteins for recognition and for ATPase/RNA helicase reaction (A. Pause and N. Sonenberg, Current Opinion in Structural Biology Vol 3, 1993, 953-959).
The DEAD box protein family is an enzyme class which is growing and which is involved in the various reactions in post transcriptional regulation of gene expression. Because of the high number of different cellular DEAD box proteins, it is to be expected that specific RNA helicases are assigned to certain classes of gene products, e.g. viral proteins, heat shock proteins, antibody and MHC proteins, receptors, RNAs etc. This specificity indicates that members of this protein family are attractive pharmacological targets for active compound development.
Two of the subclasses of the DEAD box protein family are the DEAH proteins (having one specific amino acid replacement) and the DEXH protein (having two amino acid replacements in the main motif, X being any desired amino acid) families, which also play a part in the replication, recombination, repair and expression of DNA and RNA genomes (Gorbalenya, A. E., Koonin, E. V., Dochenko, A. P., Blinov, V. M., 1989: Nucleic Acids Res. 17, 4713-4729).
The DEAD box proteins and their subfamilies are often designated “helicase superfamily II” (Koonin, E. V., Gorbalenya, A. E., 1992: FEBS 298, 6-8). This superfamily has seven highly conserved regions. Altogether, up to now over 70 members belong to this superfamily II.
The following schematic representation of the DEAD family and the DEAH and DEXH families subfamilies (Schmid, S. R., Lindner P., 1991: Molecular and Cellular Biology 11, 3463-3471) shows the similarity between the families. The structure of elF-4A, a member of a DEAD box protein, is also shown. The numbers between these regions show the distances in amino acids (AA). X is any desired AA. Where known, functions have been assigned to the ranges.
DEAD FAMILY

         ATPase A motif                         ATPase B motif
(SEQ ID NO: 19)
NH
2
------AXXXGKT-----PTRELA-----GG-----TPGR-----DEAD-----SAT-----FXXXT-----
   21-299       24-42      22-28  19-27    19-22    27-51   59-70     52-53

RGXD----HRIGRXXR------COOH
     20         24-236

eIF-4A

NH
2
------AXXXXGKT-----PTRELA-----GG-----TPGR-----DEAD-----SAT-----FINT-----
(SEQ ID NO: 20)
     75            24         22     20       20       27      62       52

RGID----HRIGRXXR------COOH
     20           41

DEAH SUBFAMILY

NH
2
-------GXXXXGKT-----RVAA-----XX-----TDGX-----DEAH-----SAT-----FXT-----
(SEQ ID NO: 21)
   245-505        22-24      29    7-8       19       28    58-61   75-84

XGXX----QRIGRXGR-------COOH
     25         313-373

DEXH SUB FAMILY

NH
2
-------XXXXXGKT-----PTRXXX-------------------DEXH-----TAT-----FXXZ-----
(SEQ ID NO: 22)
   81-1904        19-27             55-60           24-30   44-72    46-55

XXGX-----QRXGRXGR--------COOH
    38-44        155-1799
The ATPase motif (AXXXXGKT SEQ ID NO:23) is an amino-terminal conserved region and occurs in most proteins which bind nucleotides, i.e. also in other proteins which interact with DNA and RNA, such as DNAB (part of the primosome), UvrD (endonuclease), elongation factor 1 and transcription termination factor Rho (Ford M. J., Anton, I. A., Lane, D. P., 1988: Nature 332, 736-738). As used in this specification “ATPase activity” is used to mean the ability to catalyze hydrolysis of ATP. The ATPase A and ATPase B motifs function together in the enzymatic process of ATP hydrolysis.
The second conserved region is the so-called DEAD box, or DEAH, DEXH or DEXX box in other families of the helicases and nucleic acid-dependent ATPases. This region represents the ATPase B motif. In the reaction mechanism, the N-terminal aspartic acid in the DEAD box binds Mg
2+
via a water molecule (Pai, E. F., Krengel, U., Petsko, G. A., Gody, R. S., Katsch, W., Wittinghofer, A., 1990: EMBO J. 9, 2351-2359). Mg
2+
in turn forms a complex with the &bgr;- and gamma-phosphate of the nucleotide and is essential for the ATPase activity. Substitutions of the first two amino acids of the DEAD region in elF-4A prevent ATP hydrolysis and RNA helicase activity, but not ATP binding (Pause, A., Sonenberg, N., 1992: EMBO J. 11, 2643-2654). The DEAD region additionally couples RNA helicase activity to ATPase activity. The hydrolysis of ATP provides the energy needed for RNA unwinding during helicase activity.
The third region investigated is the SAT region (sometimes also TAT). As a result of mutation in this region, RNA helicase activity is suppressed, but other biochemical properties are retained (Pause A. & Sonenberg N., 1992). As used in this specification “helicase activity” is used to mean the ability to directly or indirectly catalyze the unwinding of RNA.
The farthest carboxy-terminal

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