PromFD 1.0: a computer program that predicts eukaryotic pol II promoters using strings and IMD matrices

Motivation: A large number of new DNA sequences with virtuallyunknown functions are generated as the Human Genome Projectprogresses. Therefore, it is essential to develop computer algorithmsthat can predict the functionality of DNA segments accordingto their primary sequences, including algorithms that can predictpromoters. Although several promoter-predicting algorithms areavailable, they have high false-positive detections and therate of promoter detection needs to be improved further. Results: In this research, PromFD, a computer program to recognizevertebrate RNA polymerase II promoters, has been developed.Both vertebrate promoters and non-promoter sequences are usedin the analysis. The promoters are obtained from the EukaryoticPromoter Database. Promoters are divided into a training setand a test set. Non-promoter sequences are obtained from theGenBank sequence databank, and are also divided into a trainingset and a test set. The first step is to search out, among allpossible permutations, patterns of strings 5–10 bp long,that are significantly over-represented in the promoter set.The program also searches IMD (Information Matrix Database)matrices that have a significantly higher presence in the promoterset. The results of the searches are stored in the PromFD database,and the program PromFD scores input DNA sequences accordingto their content of the database entries. PromFD predicts promoters—theirlocations and the location of potential TATA boxes, if found.The program can detect 71% of promoters in the training setwith a false-positive rate of under 1 in every 13 000 bp, and47% of promoters in the test set with a false-positive rateof under 1 in every 9800 bp. PromFD uses a new approach andits false-positive identification rate is better compared withother available promoter recognition algorithms. The sourcecode for PromFD is in the ‘c++’ language. Availability: PromFD is available for Unix platforms by anonymousftp to: beagle. colorado. edu, cd pub, get promFD.tar. A Javaversion of the program is also available for netscape 2.0, byhttp: // beagle.colorado.edu/chenq. Contact: E-mail: chenq{at}beagle.colorado.edu     

Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.     

Novel histone-like DNA-binding proteins in the nucleoid from the acidothermophillic archaebacterium Sulfolobus acidocaldarius that protect DNA against thermal denaturation

DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase T_m of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [³H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C).     

Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus

The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A gene in the archaebacterium Sulfolobus solfataricus that codes for a protein equivalent to the alpha subunits of the signal recognition particle receptor in eukaryotes

We have sequenced a gene in the archaebacterium Sulfolobus solfataricus that codes for a protein that shows sequence similarity to the alpha subunit of the signal recognition particle receptor or docking protein in eukaryotes and the product of the ftsY gene in Escherichia coli. Comparison of the Sulfolobus 'docking protein' with its eukaryotic and eubacterial counterparts showed that the region of highest sequence similarity corresponds to a GTP-binding site. The presence of this gene in archaebacteria suggests that some of the components involved in protein transport have been conserved in the three kingdoms.     

A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli.

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes.     

Polyphosphate as a source of phosphoryl group in protein modification in the archaebacterium Sulfolobus acidocaldarius

The incubation of polyphosphates with the ribosomal fraction of Sulfolobus acidocaldarius leads to the covalent attachment of phosphate to threonine residue(s) of a single 40,000 Mr protein. The hydrolysis kinetics of this protein showed that polyphosphate might be the modifying group. The reaction requires 2 mM Mn2+ ions and is time-dependent. ATP strongly inhibits the transfer of phosphate from polyphosphate, indicating that this process is catalyzed by an enzyme differing from the well-known protein kinases.     

Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein

Evidence that NSP2 plays a role in packaging and replication comes from studies on tsE(1400), a rotavirus mutant with a temperature-sensitive (ts) lesion in the NSP2 gene. Cells infected with tsE and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the ts phenotype of tsE. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of tsE NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that tsE NSP2 formed octamers which, like those formed by wild-type (wt) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg(2+) and the nonpermissive temperature caused disruption of the tsE octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with wt octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of tsE NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for wt NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of tsE NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.     

The structure of the gene for ribosomal protein L5 in the archaebacterium Sulfolobus acidocaldarius.

The gene for the ribosomal protein L5 from the archaebacterium Sulfolobus acidocaldarius has been isolated and sequenced. The gene codes for a basic protein of molecular weight 29 165 Da. This protein shows substantial similarity to the equivalent protein from other archaebacteria as well as from yeast, and considerably less similarity to the equivalent eubacterial protein. These results support the concept of the archaebacteria as a monophyletic kingdom more closely related to eukaryotes than to eubacteria.