Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1-kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40-kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2-terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer-assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Furthermore, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism; this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono- or dinucleotide-binding proteins.     

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

The structural repertoire of the human V kappa domain.

In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa-J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Single-stranded DNA binding proteins (SSBs) from prokaryotic transmissible plasmids

The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.     

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.     

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.     

The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.

The muscle regulatory proteins Myf3, Myf4, Myf5, and Myf6 share a highly conserved DNA binding and dimerization domain consisting of a cluster of basic amino acids and a potential helix-loop-helix structure. Here we demonstrate that the four human muscle-specific HLH proteins have similar DNA binding and dimerization properties. The members of this family form protein complexes of comparable stability with the ubiquitously expressed HLH proteins E12, E2-2, and E2-5 and bind to the conserved DNA sequence CANNTG designated as E-box with similar efficiency in vitro. The binding affinities of the various complexes are greatly influenced by the variable internal and flanking nucleotides of the consensus motif. Combinations of Myf proteins with one another and with lyl-1, and HLH protein from human T cells, do not bind to DNA in vitro. Our results suggest that combinatorial associations of the various tissue-specific and more widely expressed HLH factors do not result in differential recognition of DNA sequences by Myf proteins.     

ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development.

The ARID family of DNA binding proteins was first recognized approximately 5 years ago. The founding members, murine Bright and Drosophila dead ringer (Dri), were independently cloned on the basis of their ability to bind to AT-rich DNA sequences, although neither cDNA encoded a recognizable DNA binding domain. Mapping of the respective binding activities revealed a shared but previously unrecognized DNA binding domain, the consensus sequence of which extends across approximately 100 amino acids. This novel DNA binding domain was designated AT-rich interactive domain (ARID), based on the behavior of Bright and Dri. The consensus sequence occurs in 13 distinct human proteins and in proteins from all sequenced eukaryotic organisms. The majority of ARID-containing proteins were not cloned in the context of DNA binding activity, however, and their features as DNA binding proteins are only beginning to be investigated. The ARID region itself shows more diversity in structure and function than the highly conserved consensus sequence suggests. The basic structure appears to be a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of the basic six. It has also become apparent that the DNA binding activity of ARID-containing proteins is not necessarily sequence specific. What is consistent is the evidence that family members play vital roles in the regulation of development and/or tissue-specific gene expression. Inappropriate expression of ARID proteins is also increasingly implicated in human tumorigenesis. This review summarizes current knowledge about the structure and function of ARID family members, with a particular focus on the human proteins.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.     

RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.

Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.     

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.     

Intrinsically unstructured proteins and their functions

Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold. Disordered regions can be highly conserved between species in both composition and sequence and, contrary to the traditional view that protein function equates with a stable three-dimensional structure, disordered regions are often functional, in ways that we are only beginning to discover. Many disordered segments fold on binding to their biological targets (coupled folding and binding), whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.