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I. Introduction

     Having answered our questions about the identity of DNA as the genetic material, and filled in some details about is replication, we can turn to the most important question in the recent history of molecular biology: How is the information stored in DNA translated into biological action? Remember, DNA by itself is inert. It is akin to having a book that tells you the recipe for dynamite. By itself, the book is useless. In the hands of someone who knows how to read the instructions, collect the raw materials, and has the requisite technical skill, though, the information is potentially quite explosive.

II. One Gene - One Enzyme

     About seventy-five years ago, researchers were beginning to understand the nature of biological catalysis - the action of enzymes on their substrates that is the basis of most of the chemical reactions that occur in biological systems. These biochemists used auxotrophic strains of bacteria and fungi to develop a model stating that each metabolic reaction is carried out by an enzyme that has been specified by a unique gene (Figure 17.1).  Today, this is known to be overly simplistic, but it was correct enough to lead to the elucidation of the process of gene expression.

III. Gene Expression

     Think about your genetic information.  If you only had one copy of your recipe for dynamite, and any damage would be at best irreplaceable, at worst undetectable, you would want to safeguard that information.  If someone were making up a batch for you, you wouldn’t want to let her walk off with the book.  You might instead give her a xerox that you didn’t need back. In the same way, the first step in gene expression is to copy the genetic information into mRNA (transcription).  Then, the protein synthetic machinery (ribosomes, tRNA, amino acyl tRNA synthetases, amino acid biosynthetic pathways) can use it to construct the appropriate protein.

     It is easy to see how base pairing can specify the sequence of RNA on a DNA template. Less obvious is how mRNA can specify protein.  With only four RNA monomers (ACGU), the genetic word must be at least three characters long in order to specify twenty amino acids.  These codons are read one at a time by tRNA adapters that match the correct amino acid to its codon.  The whole process is carried out on the ribosome, and demands a constant supply of charged tRNAs.

Researchers used synthetic RNA polymers to deduce the genetic code (Figures 17.3, 17.4) which is broadly identical in all creatures.  With a three-letter word, 64 combinations are possible, and the code is redundant in that multiple codons specify the same amino acid.  Each codon, though, specifies only a single amino acid, so the code is not ambiguous. Note that the position of the first codon specifies the reading frame for the rest of the protein. Mutations that affect the reading frame have a large impact on the structure of the protein.

     For an overview of the process that we are about to describe, see Figure 17.23 or this equivalent from Genentech.

IV. Transcription

     In contrast to the replication of DNA, where a number of proteins are required to carry out the process, transcription involves only RNA polymerase (a multisubunit enzyme.)  The initiation of transcription by RNA polymerase is the key point of regulation for genes that are not expressed at a constant rate.  Although any other step in the process of gene expression could in principle be regulated (and many are) regulation of transcription initiation is the most efficient in terms of raw materials used.  Why make a copy just to throw it away? 

     Transcription initiation involves polymerase recognizing a promoter, opening the DNA, and synthesizing the first few bases of the RNA chain. The presence or absence of transcription factors helps the polymerase decide which genes are to be transcribed. Once initiated, transcription proceeds at about 60 bases per second and is terminated in a sequence - specific manner. See Figure 17.6.

V. Translation

     Translation is a multistep process that requires codon-to-amino-acid adapters (tRNA; Figure 17.12) and a place for these adapters to contact the mRNA chain in an organized fashion - the ribosome. tRNA molecules are short (~80 base) RNA chains that contain an anticodon bas sequence and an attachment site for the amino acid specified by that anticodon, Note that some of the degeneracy of the genetic code is due to the fact that some tRNA molecules recognize several codons, usually wobble variants.

     Ribosomes, assembled in the nucleolus of eukaryotic cells, are the structures on which protein synthesis occurs. Translation (See Figures 17.14 - 17.17) begins when a free small ribosomal subunit, with an initiator tRNA bind to the initiator codon downstream from the transcriptional start signal. The initiation complex is complete when the large ribosomal subunit binds, in a process that requires GTP hydrolysis. Elongation then proceeds with the binding of the proper aa-tRNA at the A site, peptide bond formation, and translocation. The translocation step uses one molecule of GTP, and results in a free A site for the binding of the subsequent tRNA. Translation is terminated when a release factor binds to a stop codon in the A site of the ribosome.

VI. Post-Translation

     In order to be active, many proteins require modification. They might be cleaved, transported, or assembled, or disulfide bridges or other covalent modification might take place. Proteins that are destined for specific subcellular compartments or for secretion contain localization signal sequences that are used by the protein sorting machinery to move proteins to their proper location. See Figure 17.19.

VII. RNA Processing

     In Eukaryotes, a number of additional steps are carried out between transcription and translation. mRNA 5’ ends are capped, and 3’ poly-A tails are added. Introns (see Figure 17.8) are removed by the spliceosome (although some pre-mRNAs are self-splicing), and the mature transcript is transported out of the nucleus for translation in the cytoplasm. Some of these steps provide opportunities to construct more than one protein from a single transcript.

VIII. Mutation

     Now that we understand the steps of gene expression, we can look at how changes in the sequence of DNA impact the function of proteins specified by the altered gene. Frameshift mutations are, in general, more deleterious than are point mutations.  Frameshifts may be insertions or deletions, while point mutations can be neutral, missense or nonsense.  See the Ames test (page 314) as a measure of the ability of a compound to induce back mutation in an auxotrophic bacterial strain.