Translation Start Signal Research Articles

Selection on start codons in prokaryotes and potential compensatory nucleotide substitutions

Reconstruction of the evolution of start codons in 36 groups of closely related bacterial and archaeal genomes reveals purifying selection affecting AUG codons. The AUG starts are replaced by GUG and especially UUG significantly less frequently than expected under the neutral expectation derived from the frequencies of the respective nucleotide triplet substitutions in non-coding regions and in 4-fold degenerate sites. Thus, AUG is the optimal start codon that is actively maintained by purifying selection. However, purifying selection on start codons is significantly weaker than the selection on the same codons in coding sequences, although the switches between the codons result in conservative amino acid substitutions. The only exception is the AUG to UUG switch that is strongly selected against among start codons. Selection on start codons is most pronounced in evolutionarily conserved, highly expressed genes. Mutation of the start codon to a sub-optimal form (GUG or UUG) tends to be compensated by mutations in the Shine-Dalgarno sequence towards a stronger translation initiation signal. Together, all these findings indicate that in prokaryotes, translation start signals are subject to weak but significant selection for maximization of initiation rate and, consequently, protein production.

A two-component, multimeric endolysin encoded by a single gene.

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

Clinical and mutation data in 12 patients with the clinical diagnosis of Nager syndrome

Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T>C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.

Domestication of Transposable Elements into MicroRNA Genes in Plants

Transposable elements (TE) usually take up a substantial portion of eukaryotic genome. Activities of TEs can cause genome instability or gene mutations that are harmful or even disastrous to the host. TEs also contribute to gene and genome evolution at many aspects. Part of miRNA genes in mammals have been found to derive from transposons while convincing evidences are absent for plants. We found that a considerable number of previously annotated plant miRNAs are identical or homologous to transposons (TE-MIR), which include a small number of bona fide miRNA genes that conform to generally accepted plant miRNA annotation rules, and hairpin derived siRNAs likely to be pre-evolved miRNAs. Analysis of these TE-MIRs indicate that transitions from the medium to high copy TEs into miRNA genes may undergo steps such as inverted repeat formation, sequence speciation and adaptation to miRNA biogenesis. We also identified initial target genes of the TE-MIRs, which contain homologous sequences in their CDS as consequence of cognate TE insertions. About one-third of the initial target mRNAs are supported by publicly available degradome sequencing data for TE-MIR sRNA induced cleavages. Targets of the TE-MIRs are biased to non-TE related genes indicating their penchant to acquire cellular functions during evolution. Interestingly, most of these TE insertions span boundaries between coding and non-coding sequences indicating their incorporation into CDS through alteration of splicing or translation start or stop signals. Taken together, our findings suggest that TEs in gene rich regions can form foldbacks in non-coding part of transcripts that may eventually evolve into miRNA genes or be integrated into protein coding sequences to form potential targets in a “temperate” manner. Thus, transposons may supply as resources for the evolution of miRNA-target interactions in plants.

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

NGASP--the nematode genome annotation assessment project.

BackgroundWhile the C. elegans genome is extensively annotated, relatively little information is available for other Caenorhabditis species. The nematode genome annotation assessment project (nGASP) was launched to objectively assess the accuracy of protein-coding gene prediction software in C. elegans, and to apply this knowledge to the annotation of the genomes of four additional Caenorhabditis species and other nematodes. Seventeen groups worldwide participated in nGASP, and submitted 47 prediction sets across 10 Mb of the C. elegans genome. Predictions were compared to reference gene sets consisting of confirmed or manually curated gene models from WormBase.ResultsThe most accurate gene-finders were 'combiner' algorithms, which made use of transcript- and protein-alignments and multi-genome alignments, as well as gene predictions from other gene-finders. Gene-finders that used alignments of ESTs, mRNAs and proteins came in second. There was a tie for third place between gene-finders that used multi-genome alignments and ab initio gene-finders. The median gene level sensitivity of combiners was 78% and their specificity was 42%, which is nearly the same accuracy reported for combiners in the human genome. C. elegans genes with exons of unusual hexamer content, as well as those with unusually many exons, short exons, long introns, a weak translation start signal, weak splice sites, or poorly conserved orthologs posed the greatest difficulty for gene-finders.ConclusionThis experiment establishes a baseline of gene prediction accuracy in Caenorhabditis genomes, and has guided the choice of gene-finders for the annotation of newly sequenced genomes of Caenorhabditis and other nematode species. We have created new gene sets for C. briggsae, C. remanei, C. brenneri, C. japonica, and Brugia malayi using some of the best-performing gene-finders.

Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.

Presence of ATG triplets in 5′ untranslated regions of eukaryotic cDNAs correlates with a ‘weak’ context of the start codon

The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it.

A peptide profile of the Bacillus subtilis genome

Bacillus subtilis is known to produce an abundance of small polypeptides. Several of these have antimicrobial activity and others are pheromones or extracellular factors that affect internal signal transduction systems. The completion of the B. subtilis genomic nucleotide sequence has revealed 345 small polypeptide open-reading frames (of 85 codons or less), 81% of which are of unknown function. A significant number of these reside in prophage genomes or phage-like elements where they can be organized into large operons. It is likely that many more exist in the genome of B. subtilis but are “hidden” entirely or partially within other reading frames, or possess non-conventional translation start signals and have escaped detection. The discovery of so many small polypeptide orfs (SPORFs) and the likelihood of many more pose a challenging problem for those undertaking the complete functional analysis of genes that constitute prokaryotic genomes. A survey of known and potential peptide-encoding reading frames is presented herein as an attempt to classify those that are found in the B. subtilis genome according to function inferred from homology searches and to conservation among products of other microbial genomes.

A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo

A highly specific monoclonal antibody, which recognises an epitope between amino acids (aa) 408 and 446 of EBNA-1, was found to immunoprecipitate this protein with great efficiency. By adding a 39-aa tag, the BGLF4 protein product of EBV was shown to express in the cytoplasm of transfected cells. This EBNA-1 tag could be used for the detection of specific protein expression by immunoblotting, immunofluorescence and, especially, immunoprecipitation assays. A plasmid vector encoding this EBNA-1 tag sequence was therefore designed for efficient expression both in vitro and in vivo. The efficient translational start signal of black beetle virus (BBV) was placed under the control of the SV40 or T7 promoters, and the β-globin splicing signal used to enhance the transportation of mRNA from the nucleus to the cytoplasm.

Structural analysis and transcript processing of the bovine proteolipid protein (PLP) gene.

In this study we present the complete genomic structure of the bovine PLP gene and its assignment to the long arm of the X-chromosome (BTXq2.1). We determined a total of 18,767 bp of the bovine PLP gene and compared it to the human heterolog. A very high similarity was detected between the non-coding regions, interrupted primarily by several transposable elements. A deletion of 13 bp in the vicinity to the translation start signal in the promoter of the bovine PLP gene was found. Functional studies of the 3' region showed the use of several polyadenylation signals. Three main transcripts were detected in adult cattle in the range of 3200, 2400, and 1600 nucleotides using Northern blot analysis. An additional shorter transcript was detected in the cerebrum of calves.

Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium.

The wzy/rfc gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wzy transcriptional startpoint was initially identified by primer extension. Next, wzy promoter strength in Escherichia coli K-12 was measured, and was found to be greater than that of the induced lac promoter. To define the Wzy translational startpoint, DNA including the wzy promoter and the putative first five residues of the Wzy protein was fused to the N-terminus of glutathione-S-transferase, and the fusion protein purified by affinity chromatography. N-terminal amino acid sequencing yielded the Wzy translational startpoint. Next, the Wzy protein was C-terminally tagged with the FLAG peptide, and immunoblotting of an S. typhimurium strain expressing a low-copy wzy-FLAG gene (five copies per cell) localized the intact Wzy protein in the cytoplasmic membrane of S. typhimurium cells. The Wzy protein was not well-expressed from a multi-copy wzy-FLAG+ plasmid in S. typhimurium, or in E. coli K-12.

Hormone-independent Transcriptional Activation and Coactivator Binding by Novel Orphan Nuclear Receptor ERR3

Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

Transposition of IS117 of Streptomyces coelicolor A3(2) in Mycobacterium smegmatis

Derivatives of IS117, the Streptomyces coelicolor A3(2) 2.6 kb minicircle, transpose efficiently in Mycobacterium smegmatis, targeting chromosomal sites resembling translation start signals. Two IS117 derivatives, pIJ4696 and pIJ4697, containing a Streptomyces hygromycin-resistance gene in opposite orientations were introduced into M. smegmatis by electroporation and found to integrate into one of three specific sites. Integrations at sites A and B were frequent while integration at site C was observed only once. Only one site was occupied in each transformant. Sites A and B had either single or tandem integrations. PFGE analysis located these sites on different genomic Asel fragments. The sequences of the chromosome-IS117 junctions confirmed that integration was via the same IS117 attachment site as in Streptomyces, that there was no target site duplication, and that the orientation of IS117 at each site was fixed. In contrast to the situation in Streptomyces lividans, no deletions were created by the transposition and no circular forms could be detected. Comparison of the three M. smegmatis chromosomal 15117 target sites (attB) with known primary and secondary S. lividans attB sites showed that only a 2 bp 'AG' sequence at the crossover point was conserved. Dividing the attB sites into two groups produced two longer consensus target sites, GtcAAGg and gCCGATAGg. Most of the IS117 target sites resemble translational start sites, and site C resembles strongly the amino-terminal sequence of a Mycobacterium tuberculosis aminopeptidase. The level of hygromycin resistance in the transformants was high and independent of the site of integration, the number of copies integrated, or the orientation of the hyg gene. pIJ4696 at all three sites was stable in M. smegmatis in the absence of selection for at least 60 cell divisions. pIJ4696, pIJ4697 and other IS117 derivatives are promising vectors for the stable, integrative cloning of genes in M. smegmatis.

Characterization of the nucleoside triphosphate phosphohydrolase I gene from the Choristoneura fumiferana entomopoxvirus

Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene ( nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus (CfEPV) has been located in the viral genome, cloned and characterized. It has an open reading frame of 1941 nucleotides, potentially encoding a protein with a predicted molecular mass of 76.04 kDa and a pI of 8.83. It has a TAAATG motif where the trinucleotide ATG represents the translational start signal an AT-rich (88%) sequence and an early transcription termination signal (TTTTTAT) upstream of the ATG codon. Northern blot analysis of mRNA from infected larvae showed that a single 4.0 kb transcript which appeared late at day 20 post infection (p.i.) and its transcription continued till day 37 p.i.. Primer extension experiments suggested that the main transcripts started at 15 bases upstream of AUG codon. NPH I homologues have been found in the genomes of other entomopoxviruses and vertebrate poxviruses. Alignment of their amino acid sequences suggested three conserved domains, two of which are considered as ATP binding domains. The most similar homologue is from the closely related entomopoxvirus, Choristoneura biennis EPV (CbEPV) where 98.2% of nucleotide and 97.2% of amino acid identities are observed, respectively. A single nucleotide difference in CfEPV nph I was sufficient to distinguish it from CbEPV by PCR amplification and digestion with a restriction enzyme.

The Chromosome Location of the Human Homolog of the Mouse Mammary Tumor-Associated GeneINT6and Its Status in Human Breast Carcinomas

TheINT6gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog ofINT6is located on chromosome region 8q22–q23. A processedINT6pseudogene is located on chromosome 6q.INT6is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affectingINT6.Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 ofINT6.Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked toINT6.

Generation of new DNA binding specificity by truncation of the type IC EcoDXXI hsdS gene.

The hsdS subunit of a type IC restriction-modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half-sites separated by a non-specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half-site. We have shown previously that the 5' half of hsdS can code for a functional substitute of the full-length hsdS. Here we demonstrate that the 3' half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full-length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5' hsdS mutant, the 3' mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co-expression of the 5' hsdS mutant and the 3' hsdS mutant along with hsdM regenerates the wild-type methylation specificity. Thus, there is a free assortment of subunits in the cell.

The herpes simplex virus gene UL26 proteinase in the presence of the UL26.5 gene product promotes the formation of scaffold-like structures.

The herpes simplex virus type 1 (HSV-1) polypeptides encoded by genes UL26 and UL26.5 are thought to form a scaffold around which the capsid shell assembles. The UL26 gene specifies a proteinase that cleaves both itself and the UL26.5 gene product. To study the structure and function of the UL26 and UL26.5 gene products, the proteins were expressed in cells infected with recombinant baculoviruses containing the genes under the control of the polyhedrin promoter. Both polypeptides were made in large amounts, approaching the levels of polyhedrin protein expressed in wild-type baculovirus. The UL26 polypeptide behaved in a similar manner to the protein made in HSV-1-infected cells, cleaving itself rapidly into the capsid proteins VP21 and VP24 and converting the UL26.5 product more slowly into the capsid protein VP22a. The results of immunoblot analysis using antisera specific for the amino-terminal region of the UL26 polypeptide suggested that both the first and second ATGs in the UL26 open reading frame were recognized as translational start signals but the first ATG was the preferred initiation codon as is the case in HSV-1-infected cells. Electron microscopic examination of thin section preparations of cells infected with both the UL26.5- and UL26-recombinant baculoviruses revealed the presence of large numbers of small spherical particles, often arranged in a semi-crystalline array. These clusters of scaffold-like particles were not present in cells infected with UL26-recombinant baculovirus but were observed occasionally in UL26.5-recombinant baculovirus-infected cells. The results suggest that the proteinase, in the absence of other HSV capsid proteins, stimulates the formation of large numbers of scaffold-like particles present either as semi-crystalline arrays or as dispersed structures.

The role of RNA editing in conservation of start codons in chloroplast genomes

Open reading frames (ORFs), encoded by the plastid genomes of tobacco, liverwort, rice and maize were aligned with a view to studying the conservation of translational start and stop codons created by RNA editing of homologous genes. It became evident that most of the homologous ORFs have conserved translation start and stop signals at the gene level. However, some of the ORFs show differences with respect to extensions of their 3' and 5' terminal regions. For example, the proposed N-termini of the ndhD-encoded peptides from different plant species are very variable in length and amino-acid composition. Sequence analysis of ndhD and the corresponding cDNA shows that editing of an ACG triplet in tobacco, spinach and snapdragon leads to the creation of an AUG codon, corresponding to the start codon in other species. Conservation of translational start codons of plastome-encoded genes can, therefore, be achieved by editing of transcripts, and the definition of plastome-encoded ORFs must take potential editing events into consideration.

Analysis of the Bacillus subtilis Bacteriophages SPP1 and SF6 Gene 1 Product: A Protein Involved in the Initiation of Headful Packaging

Gene 1 product (G1P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and ρ15 is essential for DNA maturation and packaging. A DNA segment containing gene 1 of phage SF6 or ρ15 origin was cloned and sequenced. SF6 and ρ15 G1P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G1P of SPP1. The G1P of all three phages contains three conserved segments (I, II, and III). Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted. G1P of both SPP1 and SF6 origin was purified. SPP1 G1P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa. The 13-kDa polypeptide results from a translation start signal within gene 1, and hence was termed SPP1 G1P*. G1P of both SPP1 and SF6 binds specifically to a pac -containing DNA fragment, whereas G1P*, which lacks segment I, does not. Chimeric G1P protiens were obtained by domain swapping between gene 1 of SPP1 and SF6. The results presented here suggest that the G1P DNA binding motif lies in segment I and the major determinant for G1P::G1P interaction might lie in segment II. Segment III and the extended C-terminal part of SPP1 G1P are dispensable. The G1P::G2P interacting region remains uncharacterized.