The Central Dogma

By the fall of 1953, the working hypothesis was adopted that chromosomal DNA functions as the template for RNA molecules, which subsequently move to the cytoplasm, where they determine the arrangement of amino acids within proteins. En 1956, Francis Crick referred to this pathway for the flow of genetic information as the central dogma.

Duplication

Transcription Translation DNA -> RNA —* Protein

Here the arrows indicate the directions proposed for the transfer of genetic information. The arrow encircling DNA signifies that DNA is the template for its self-replication. The arrow between DNA and RNA indicates that RNA synthesis (transcription) is directed by a DNA template. Correspondingly, the synthesis of proteins (translation) is directed by an RNA template. Most importantly, the last two arrows were presented as unidirectional; that is, RNA sequences are never determined by protein templates, nor was DNA then imagined ever to be made on RNA templates. That proteins never serve as templates for RNA has stood the test of time. However, as we will see in Chapter 11, RNA chains sometimes do act as templates for DNA chains of complementary sequence. Such reversals of the normal flow of information are very rare events compared with the enormous number of RNA molecules made on DNA templates. Thus, the central dogma as originally proclaimed approximately 50 years ago still remains essentially valid.

The Adaptor Hypothesis of Crick

At first it seemed simplest to believe that the RNA templates for protein synthesis were folded up to create cavities on their outer surfaces specific for the 20 different amino acids. The cavities would be so

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32 Nui leir.: Adds Convey Gefir.'fii. Information

FIGURE 2-13 Electron micrograph of ribosomes attached to the endoplasmic reticulum. This electron micrograph (105,000k) shows a portion ot a pancfeatic celf. Die upper fight portirm shows a portion of the mitochondrion and the tower left shows a lafge number of ribosomes attached to the endoplasmic reticulum. Some ribosomes exist free in the cytoplasm, others are attached to the membra nous endoplasmic reticulum (Source: Courtesy of (CR Porter )

FIGURE 2-13 Electron micrograph of ribosomes attached to the endoplasmic reticulum. This electron micrograph (105,000k) shows a portion ot a pancfeatic celf. Die upper fight portirm shows a portion of the mitochondrion and the tower left shows a lafge number of ribosomes attached to the endoplasmic reticulum. Some ribosomes exist free in the cytoplasm, others are attached to the membra nous endoplasmic reticulum (Source: Courtesy of (CR Porter )

shaped that only one given amino acid would fit, and in this way RNA would provide the information to order amino acids during protein synthesis. By 1955, however, Crick became disenchanted with this conventional wisdom, arguing that it would never work. In the first place, the specific chemical groups on the four liases of KNA {A, U, G, and C) should mostly interact with water-soluble groups. Yet, the specific side groups of many amino acids (for example, leucine, valine, and phenylalanine) strongly prefer interactions with water-insoluble (hydrophobic) groups. In the second plane, even if somehow RNA could be folded so as to display some hydrophobic surfaces, it seemed at the time unlikely that an RNA template would be used to discriminate accurately between chemically very similar amino acids like glycine and alanine or valine and isoleucme, both pairs differing only by the presence of single methyl (CHj) groups. Crick thus proposed that prior to incorporation into proteins, amino acids are first attached to specific adaptor molecules, which in turn possess unique surfaces that can bind specifically to bases on the RNA templates.

The Test-Tube Synthesis of Proteins

The discovery of how proteins are synthesized required the development of cell-free extracts capable of carrying on the essential synthetic steps. These were first effectively developed beginning in 1953 by Paul C. Zarnecnik arid his collaborators. Key to their success were the recently available radioactively--tagged amino acids, which they used to mark the trace amounts of newly made proteins, as well as high-quality, easy-to-use, preparative ultracentrifuges for fractionation oftheir cellular extracts. Early on, the cellular site of protein synthesis was pinpointed to be the ribosomes, small RNA-contaming particles in the cytoplasm of all cells actively engaged in protein synthesis (Figure 2-13).

Several years later, Zarnecnik, by then collaborating with Mahlon B, Hnagland, went on to make the seminal discovery that prior to their incorporation into proteins, amino acids are first attached to what we now call transfer RNA (tRNA) molecules by a class of enzymes called aminoacyl synthetases. Transfer RNA accounts for some 10% of all cellular RNA (Figure 2-14).

To nearly everyone except Crick, this discovery was totally unexpected, Ke had, of course, previously speculated that his proposed "adaptors" might be short RNA chains, since their bases would be able tn base-pair with appropriate groups on the RNA molecules that served as the templates for protein synthesis. As we shall relate later in greater detail (Chapter 14}, the transfer RNA molecules of Zarnecnik and Hoagland are in feet the adaptor molecules postulated by Crick. Each transfer RNA contains a sequence of adjacent bases (the anti-codon) that bind specifically during protein synthesis to successive groups of bases (codons) along the RNA templates.

The Paradox of the Nonspecific-Appearing Ribosomes

About 85% of cellular RNA is found in ribosomes, and since its absolute amount ts greatly increased in cells engaged in large-scale protein synthesis (for example, pancreas and liver cells and rapidly growing bacteria), ribosomal RNA (rRNA) was initially thought to be the template for ordering amino acids. But once the ribosomes of E. coli were carefully analyzed, several disquieting features emerged. First, all E. coli ribosomes, as well as those from all other organisms.

are composed of two unequally-sized subunits, each containing RNA, that either stick together or fall apart in a reversible manner, depending on the surrounding ion concentration. Second, all the rRNA chains within the small subunits are of similar chain lengths (about 1,500 bases in E. coli), as are the rRNA chains of the large subunits (about 3,000 bases). Third, the base composition of both the small and large rRNA chains is approximately the same (high in C and Q in all known bacteria, plants, and animals, despite wide variations in the AT/GC ratios of their respective DNA. This was not to be expected if the rRNA chains were in fact a large collection of different RNA templates made of a large number of different genes. Thus, neither the small nor large class of rRNA had the feel of template RNA.

Discovery of Messenger RNA (mRNA)

Cells infected with phage T4 provided the ideal system to find the true template. Following infection by this virus, cells stop synthesizing E. coli RNA; the only RNA synthesized is transcribed off the T4 DNA. Most strikingly, not only does T4 RNA have a base composition very similar to T4 DNA, but it does not bind to the ribosomal proteins that normally associate with rRNA to form ribosomes. Instead, after first attaching to previously existing ribosomes, T4 RNA moves across their surface to bring its bases into positions where they can bind to the appropriate tRNA-arnino acid precursors for protein synthesis (Figure 2-15), in so acting, T4 RNA orders the amino acids and is thus the long-sought-for RNA template for protein synthesis. Becaustj it curries the information from DNA to the ribosomal sites of protein synthesis, it is called messenger RNA (mRNA). The observation of T4 RNA binding to E. coli ribosomes, first made in the spring of 1960, was soon followed with evidence for a separate messenger class of RNA within uninfected E. coli cells, thereby definitively ruling out a template role for any rRNA, Instead, in ways thai we shall discuss more extensively in Chapter 14, the rRNA components of ribosomes, together with some 50 different ribosomal proteins that bind to them, serve as the factories for protein synthesis, functioning to bring together the tRNA-amino acid precursors into positions where they can read off the information provided by the messenger RNA templates.

Only some 4% of total cellular RNA is mRNA. This RNA shows the expected large variations in length, depending on the polypeptides for which they code. Hence, it is easy to understand why mRNA was first overlooked. Because only a small segment of mRNA is attached at a given moment to a ribosome, a single mRNA molecule can simultaneously be read by several ribosomes. Most ribosomes are found as parts of polyribosomes (groups of ribosomes translating the same mRNA), which can include more than 50 members (Figure 2-1G).

Enzymatic Synthesis of RNA upon DNA Templates

As messenger RNA was being discovered, the first of the enzymes that transcribe RNA off DNA templates was being independently isolated in the labs of biochemists Jerard Hurwitz and Sam B. Weiss. Called RNA polymerases, these enzymes function only in the presence of DNA, which serves as the template upon which single-stranded RNA chains are made, and use the nucleotides ATP, CTP, CTP, and DTP as precursors

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