This strategy works very well for compact genomes, where intron sequences are rare and ORFs often extend for many hundreds of amino acids. However, in many animals and plants, the average exon size is 150–200 nucleotide pairs (see Figure 6–31) and additional information is usually required to unambiguously locate all the exons of a gene. Although it is possible to search genomes for splicing signals and other features that help to identify exons (codon bias, for example), one of the most powerful methods is simply to sequence the total RNA produced from the genome in living cells. As can be seen in Figure 7–3, this RNA-seq information, when mapped onto the genome sequence, can be used to accurately locate all the introns and exons of even complex genes. By sequencing total RNA from different cell types, it is also possible to identify cases of alternative splicing (see Figure 6–26).
RNA-seq also identifies noncoding RNAs produced by a genome. Although the function of some of them can be readily recognized (tRNAs or snoRNAs, for example), many have unknown functions and still others probably have no function at all (discussed in Chapter 7, pp. 429–436). The existence of the many noncoding RNAs and our relative ignorance of their function is the main reason that we know only the approximate number of genes in the human genome.
But even for protein-coding genes that have been unambiguously identified, we still have much to learn. Thousands of genomes have been sequenced, and we know from comparative genomics that many organisms share the same basic set of proteins. However, the functions of a very large number of identified proteins remain unknown. Depending on the organism, approximately one-third of the proteins encoded by a sequenced genome do not clearly resemble any protein that has been studied biochemically. This observation underscores a limitation of the emerging field of genomics: although comparative analysis of genomes reveals a great deal of information about the relationships between genes and organisms, it often does not provide immediate information about how these genes function, or what roles they have in the physiology of an organism. Comparison of the full
Figure 8–40 Finding the regions in a DNA sequence that encode a protein.
(A) Any region of the DNA sequence can, in principle, code for six different amino acid sequences, because any one of three different reading frames can be used to interpret the nucleotide sequence on each strand. Note that a nucleotide sequence is always read in the 5ʹ-to-3ʹ direction and encodes a polypeptide from the N-terminus to the C-terminus. For a random nucleotide sequence read in a particular frame, a stop signal for protein synthesis is encountered, on average, about once every 20 amino acids. In this sample sequence of 48 base pairs, each such signal (stop codon) is colored blue, and only reading frame 2 lacks a stop signal. (B) Search of a 1700-base-pair DNA sequence for a possible protein-encoding sequence. The information is displayed as in (A), with each stop signal for protein synthesis denoted by a blue line. In addition, all of the regions between possible start and stop signals for protein synthesis (see pp. 347–349) are displayed as red bars. Only reading frame 1 actually encodes a protein, which is 475 amino acid residues long.
gene complement of several thermophilic bacteria, for example, does not reveal why these bacteria thrive at temperatures exceeding 70°C. And examination of the genome of the incredibly radioresistant bacterium Deinococcus radiodurans does not explain how this organism can survive a blast of radiation that can shatter glass. Further biochemical and genetic studies, like those described in the other sections of this chapter, are required to determine how genes, and the proteins they produce, function in the context of living organisms.
DNA Cloning Allows Any Protein to be Produced in Large Amounts