To some extent, the choice of which database to search will depend on which databases are provided by the site that runs the search algorithms. Most sites contain a selection of the most popular databases, such as GenBank for DNA sequences, SWISS-PROT for annotated protein sequences, TrEMBL, a translated EMBL DNA-sequence database, and PDB, a database of protein structures with
103 sequences (see Chapter 3). Some sites also provide access to expressed sequence
tag (EST) databases, such as dbEST, and genome-sequence databases from some of the fully sequenced genomes
In general, a first pass should be run on a generic protein- or nucleic acid sequence database. You can also carry out a search on the PDB to see if your query sequence has a homolog with known structure. More specific searches can be performed to answer particular questions. For example, if it is suspected that the query sequence belongs to a family of immune-system proteins, the search could be carried out on the Kabat database, which contains sequences of immunological interest. If the sequence orig- inates from a mouse, you may want to know if a homolog exists in the rat, Drosophila, or human genomes, and should therefore search the databases containing sequences from the appropriate species. You also need to check that you are searching a database that is up to date; sites such as those at NCBI and EBI are regularly updated.
If no match is found to the query sequence, it does not necessarily mean that there is no homolog in the databases, just that the similarity is too weak to be picked up by existing techniques. Techniques are continually being improved and the amount of sequence data continues to increase; you should therefore periodically resubmit your sequence.
Many other sequence-related databases can usefully be searched and provide addi- tional information. For example the Sequences Annotated by Structure (SAS) server is a collection of programs and data that can help identify a protein sequence by using structural features that are the result of sequence searches of annotated PDB sequences. Residues in the sequences of known structures are colored according to selected structural properties, such as residue similarity, and are displayed using a Web browser. SAS will perform a FASTA alignment of the query sequence against sequences in the PDB database and return a multiple alignment of all hits. Each of the hits is annotated with structural and functional features. That information can be used to annotate the unknown protein sequence. Further links are provided to the separate PDB files. Databases such as Clusters of Orthologous Groups (COGs) and UniGene can help in gene discovery, gene-mapping projects, and large-scale expres- sion analysis. Sites such as Ensembl provide convenient access for gene searches in many different annotated eukaryotic genomes and useful associated information.
4.8 Protein Sequence Motifs or Patterns
If the similarity between an unknown sequence and a sequence of known function is limited to a few critical residues, then standard alignment searches using BLAST or FASTA against general sequence databases such as GenBank, dbEST, or SWISS- PROT will fail to pick up this relationship. What is required is a method of searching
for the occurrence of short sequence patterns, or motifs (see Flow Diagram 4.4). A
motif, in general, is any conserved element of a sequence alignment, whether composed of a short sequence of contiguous residues or a more distributed pattern. Functionally related sequences will share similar distribution patterns of critical functional residues that are not necessarily contiguous. For example, conserved amino acid residues comprising an enzyme's active site may be distant from each other in the protein sequence but will still occur in a recognizable pattern because of the constraints imposed by the requirement for them to come together in a particular spatial configuration to form the active site in the three- dimensional structure.
There are three different types of activity associated with pattern searching. A query
sequence can be searched for patterns (from a patterns database) that could help suggest functional activity. A sequence database can be searched with a specific pattern, for example to determine how many gene products in a genome have a
specific function. Lastly, we may want to define a new pattern specific for a selected set of sequences.
In searches with new sequences, the whole database is searched and expert knowl- edge, such as the known function of a homologous protein, is then used to extrap- olate the function of the new sequence. In contrast, when new patterns and motifs are used to search a database, the expert knowledge is needed right at the begin- ning to construct the motifs that are intended to identify the specific function or any other physicochemical property.
Pattern and motif searches are mostly used with protein sequences rather than nucleotide sequences, as the greater number of different amino acids makes protein patterns more efficient in discriminating truly significant hits. In addition, many of the patterns identify biological function, which is mediated at the protein level. There are, however, particular problems in DNA- and genome-sequence analysis where searching for motifs is useful (see Chapters 9 and 10).
Flow Diagram 4.4
The key concepts introduced in this and the following two sections are that sequence patterns can be very useful in identifying protein function and that special pattern databases and search programs have been designed to assist in identifying patterns in a query sequence.