Biochemistry - Chemical Reactions in Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Transcription of RNA Molecules
Transfer RNA (tRNA)

The best-studied form of RNA is the small 4S tRNA molecule with a Molecular Weight of approximately 26,000, consisting of 75±5 NUCLEOTIDES (Figs. 2-24 and 15-8). Bacterial and eukaryotic tRNAs appear to be very similar in size and core structural characteristics. The existence of "adapters" required to deliver Amino Acids to the correct positions on the mRNA template chain was predicted even before tRNA was discovered. It was hypothesized that an adapter molecule must contain a nucleotide sequence forming an anticodon, which would align precisely opposite the corresponding codon in a specific binding site of the protein-synthesizing system. As we now know, tRNA molecules indeed possess these predicted properties, though investigating their chemical Structure revealed many surprises. First, tRNA molecules turned out to be longer than strictly necessary to function merely as adapters, and second, many of their constituent bases are heavily modified compared to their canonical precursors [60].

Another striking feature of tRNA architecture is that their anticodons can contain non-standard bases. For example, the anticodons of certain tRNAs incorporate hypoxanthine (whose nucleoside is inosine).

Fig. 15-8A depicts the classical cloverleaf representation of a tRNA molecule. The three-dimensional structure of tRNA, independently determined by two research groups [61, 62], is illustrated schematically in Fig. 15-8B.

Let us examine the four hydrogen-bonded stems formed by complementary nucleotide pairs (Fig. 15-8). One of these stems terminates in the acceptor end, which is the site of amino acid attachment. The acceptor end binds and carries the activated amino acid generated according to equation (11-2). The other three stems terminate in loops that typically contain a high proportion of modified bases. The dihydrouridine loop, for instance, contains varying amounts of 5,6-dihydrouridine at different positions. The anticodon loop always houses the anticodon, located at the end of the cloverleaf directly opposite the acceptor end. On the 5' side of the anticodon, there is invariably a U (circled in the figure), followed by another pyrimidine nucleotide. On the 3' side of the anticodon, one typically finds a so-called "supermodified" nucleotide. The TφC loop contains a specific conserved nucleotide sequence that gives it its name. In Bacteria, the TφC sequence has been found in all functional protein-synthesis tRNAs studied; however, in eukaryotic initiator tRNA molecules, this sequence may be replaced by UCG [63—65].

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FIG. 15-8. Top: Schematic representation of Yeast phenylalanine tRNA in its cloverleaf conformation. Dots indicate Hydrogen Bonds (two or three). Nucleotides common to nearly all tRNAs are circled, along with other universal structural features. The lower part of the diagram illustrates how the anticodon interacts with an mRNA codon. Bottom: Three-dimensional structure of yeast phenylalanine tRNA [61].

Genes encoding tRNA molecules in both bacteria and mammals are organized into clusters—groups that are cotranscribed into large precursor RNA molecules often containing more than one type of tRNA [66—69]. Maturation of functional tRNAs from these precursors through "cutting and trimming" requires at least three distinct Nucleases [57, 66, 69].

For instance, in the case of E. coli Tyrosine tRNA¹⁾, the direct precursor molecule (Fig. 15-9) contains 129 nucleotides—44 more than the mature tRNA. Of these extra nucleotides, 41 are located at the 5' end and three at the 3' end. A specific nuclease (RNase P) makes a single endonucleolytic cut in the precursor, cleaving off the 41-nucleotide fragment from the 5' end. Another nuclease (RNase PIII) then removes the three-nucleotide fragment from the opposite end [66]. The excised fragments are subsequently degraded further by auxiliary Enzymes [70].

A major milestone in chemical biology was the total synthesis of a double-stranded DNA fragment encoding E. coli tyrosine tRNA [71] and its precursor [71a], achieved by Khorana and his colleagues. These researchers subsequently extended their work to include the METABOLISM/31.html">Transcription termination region located beyond the sequence encoding the CCA end of the tRNA. They determined The sequence of 23 DNA nucleotides downstream of the CCA-coding region [72, 73] (Fig. 15-9). Two notable features emerge here. First, There is a region of twofold rotational Symmetry (indicated by vertical lines and a central dot in Fig. 15-9) that likely acts as a termination signal. Second, There are two regions containing short inverted nucleotide repeats, such as TGAAGT. Whether these particular segments function as genetic signals remains to be seen²⁾.

A 29-nucleotide region immediately preceding the tyrosine tRNA Gene contains the operator sequence, which has not yet been fully characterized [75].

In addition to precursor trimming, the biogenesis of mature tRNAs involves extensive modifications of purine and pyrimidine bases [64]. There are sixty or more known enzymatic reactions responsible for these modifications, and their number and extent vary widely across species. The chemical structures of several modified bases are illustrated in Fig. 15-10. Uridine serves as a clear example of the diverse types of modifications possible. One of the most common is methylation, which can occur on either the base ring or the 2'-hydroxyl group of the ribose sugar. Methylation of uridine at position 5 yields ribothymidine. Cytidine can also be modified at the same position. Reduction of the double bond between C-5 and C-6 produces dihydrouridine. Replacing the oxygen atom at position 4 with sulfur yields 4-thiouridine. Figure 15-10 also highlights potential methylation sites on guanosine. The symbol m is frequently used to denote methylation of nucleic acid bases (with dimethylation designated as m²⁾.

¹⁾ A minor tyrosine tRNA encoded by the suppressor gene supF (Section D, 6).

²⁾ Double-stranded DNA regions possessing twofold symmetry are often referred to as "palindromes" (Chapter 2, Section D, 11). Unfortunately, Kornberg [73] and other authors have also applied this term to adjacent inverted sequences in single-stranded DNA. Although such a palindrome might theoretically read the same in both directions, no enzymatic machinery moving along a double-stranded (or even single-stranded) DNA molecule can actually read it identically in both directions.

FIG. 15-9. Putative Introduction/11.html">Secondary structure of the E. coli tyrosine tRNA precursor. Nucleotides that occur in modified forms within the mature tRNA are depicted with their respective modifications (Schaeffer K. R., Altman S., Söll D., PNAS, 70, 3626—3630, 1973). A portion of the gene sequence downstream of the CCA-terminating region is also shown. Note the region of twofold rotational symmetry (marked by vertical lines and a dot) and the two inverted repeat sequences.

FIG. 15-10. Chemical structures of selected modified nucleosides found in tRNA molecules. Methylation sites are indicated by the letter m.

The conversion of uridine to pseudouridine (ψ) is of particular interest. Pseudouridine is known to form via the rearrangement of uridine within the primary transcript, although the exact chemical mechanism remains unclear. Crucially, pseudouridine—like uracil—can base-pair with adenine. The base designated as Y is a heavily modified guanine. Figure 15-10 also displays two "supermodified" adenosines. At the 3' ends of anticodons that pair with codons beginning with U, one finds N⁶-isopentenyladenosine. Interestingly, in plants, this very compound Functions as a hormone known as a cytokinin (Chapter 16, Section A, 3).

Another heavily modified purine, threonylcarbamoyladenosine, is typically adjacent to the anticodons that interact with codons beginning with A. While the precise Physiological Role of these heavily modified bases remains elusive, they appear essential for proper tRNA binding to Ribosomes. The specific enzyme catalyzing The transfer of the isopentenyl group from isopentenyl pyrophosphate has been isolated in pure form (Chapter 12, Section 3) [76].

An intriguing aspect of tRNA metabolism that we have not yet discussed involves the 3'-terminal CCA trinucleotide sequence. This readily cleaved and resynthesized tail is a universal feature of all tRNA molecules. The turnover rate of this sequence is high enough to process roughly 20% of the cellular tRNA pool between Cell divisions, yet it is significantly slower than the rate at which tRNAs participate in Protein Synthesis. Thus, this turnover process does not appear to be directly coupled to peptide bond formation.