The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.

Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA.

Digging for dead genes: an analysis of the characteristics of the pseudogene population in the Caenorhabditis elegans genome

Pseudogenes are non-functioning copies of genes in genomic DNA, which may either result from reverse transcription from an mRNA transcript (processed pseudogenes) or from gene duplication and subsequent disablement (non-processed pseudogenes). As pseudogenes are apparently ‘dead’, they usually have a variety of obvious disablements (e.g., insertions, deletions, frameshifts and truncations) relative to their functioning homologs. We have derived an initial estimate of the size, distribution and characteristics of the pseudogene population in the Caenorhabditis elegans genome, performing a survey in ‘molecular archaeology’. Corresponding to the 18 576 annotated proteins in the worm (i.e., in Wormpep18), we have found an estimated total of 2168 pseudogenes, about one for every eight genes. Few of these appear to be processed. Details of our pseudogene assignments are available from http://bioinfo.mbb.yale.edu/genome/worm/pseudogene. The population of pseudogenes differs significantly from that of genes in a number of respects: (i) pseudogenes are distributed unevenly across the genome relative to genes, with a disproportionate number on chromosome IV; (ii) the density of pseudogenes is higher on the arms of the chromosomes; (iii) the amino acid composition of pseudogenes is midway between that of genes and (translations of) random intergenic DNA, with enrichment of Phe, Ile, Leu and Lys, and depletion of Asp, Ala, Glu and Gly relative to the worm proteome; and (iv) the most common protein folds and families differ somewhat between genes and pseudogenes—whereas the most common fold found in the worm proteome is the immunoglobulin fold and the most common ‘pseudofold’ is the C-type lectin. In addition, the size of a gene family bears little overall relationship to the size of its corresponding pseudogene complement, indicating a highly dynamic genome. There are in fact a number of families associated with large populations of pseudogenes. For example, one family of seven-transmembrane receptors (represented by gene B0334.7) has one pseudogene for every four genes, and another uncharacterized family (represented by gene B0403.1) is approximately two-thirds pseudogenic. Furthermore, over a hundred apparent pseudogenic fragments do not have any obvious homologs in the worm.

Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds1

Semipermeable cell walls or apoplastic “membranes” have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (β-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by β-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.