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Chapter 2 : Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene

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Abstract:

Biochemical studies of protein synthesis in vitro got under way in the early 1950s. The fundamental concept arose that the specificity in protein synthesis was primarily governed by the loading of every amino acid onto a "cognate" soluble RNA by an enzyme specific for that amino acid. The RNAs involved began to be known as transfer RNAs (tRNAs), and the activating enzymes came to be known as aminoacyl-tRNA synthetases. With the elucidation of the genetic code in DNA, "the code in tRNA" emerged as a central problem in the molecular biology of protein biosynthesis, and structure and function in tRNA became the focus of attention in many laboratories. In the tRNA field, Abelson and Miller and their colleagues have carried out studies on aminoacylation specificity of tRNAs in vivo, using synthetic genes for suppressor tRNAs. This approach, together with RNA synthesis, for investigation of the sequence-dependent aminoacylation of RNA oligonucleotides in vitro has proved most useful for studying aminoacyl tRNA synthetases-tRNA recognitions in vivo. Totally synthetic genes lend themselves well to systematic mutagenesis by the principle of fragment (cassette) replacement. Structure-function studies on integral membrane proteins such as bacteriorhodopsin, sensory rhodopsin, and the vertebrate photoreceptor rhodopsin have used exclusively the synthetic gene approach. A large number of chimeric genes of visual color pigments were synthesized by Oprian and colleagues for precise studies of spectral tuning in vision.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2

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DNA Polymerase I
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DNA Synthesis
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Integral Membrane Proteins
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Figures

Image of Figure 1
Figure 1

Cloverleaf secondary structure model for yeast alanine-tRNA, originally proposed by Holley in 1965 ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 2
Figure 2

Deoxyribo-icosanucleotide segments corresponding to nucleotide sequences 21 to 50 of gene for yeast alanine tRNA. The two icosanucleotides are complementary to each other through halves of their length in the Watson-Crick antiparallel manner. Reprinted from reference with permission.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 3
Figure 3

Chemically synthesized deoxyribo-icosanucleotides corresponding to sequences 21 to 50 of yeast alanine tRNA as described in Fig. 2 . The nona-, hepta-, and penta-nucleotides (Nona-I, Hepta-I, and Penta-I) similarly contain sequences complementary to nucleotides 41 to 49 or less and have polarity opposite to that of the tRNA. The deoxyribopolynucleotides (Icosa-II, Nona-II, Hepta-II, Penta-II, and Tetra-II) are segments, complements of the complement, and therefore contain the same sequences and polarity as the tRNA itself. P represents the 5′-phosphate end group. Reprinted from reference with permission.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 4
Figure 4

Total plan for synthesis of yeast alanine tRNA structural gene. The chemically synthesized segments are shown by horizontal brackets, with the serial number of the segment in parentheses inserted into brackets. Seventeen segments (including 10′ and 12′) varying in chain length from penta- to icosa-nucleotides were synthesized. Reprinted from reference with permission.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 5
Figure 5

Primary nucleotide sequence of an tyrosine tRNA precursor. The sequence is shown with the standard cloverleaf structure for the tRNA portion and the possible hairpin at the 5′ end (reprinted with permission from [ ]).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 6
Figure 6

Linear arrangement of different parts of tyrosine-suppressor tRNA gene as originally envisaged (1970). The gene, one of the two duplicate genes in tandem, is located at 25 min on the genetic map. The suppressor gene arises by single-nucleotide change in the anticodon of one of the two tyrosine genes in tandem. Reprinted with permission ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 7
Figure 7

(A) Illustration shows nucleotide sequence in promoter region of tyrosine-suppressor tRNA gene, the point of initiation of transcription, and the direction of transcription into structural gene at right. The elements of twofold symmetry in the sequence are shown in the boxes, their correspondence being indicated by arrows. (B) Plan for total synthesis of promoter region of tyrosine-suppressor tRNA gene. Included are the 51 nucleotide base pairs in the promoter region plus one C-G base pair plus single-stranded A-A-T-T sequence at the 5′ end. The A-A-T-T sequence forms the recognition sequence for the RI restriction enzyme. The 10 segments (PI to P10) to be synthesized are indicated by horizontal brackets. Reprinted with permission ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 8
Figure 8

Nucleotide sequence in double-stranded form. Note palindromic symmetry downstream from C-C-A end of tyrosine-suppressor tRNA. Reprinted with permission ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 9
Figure 9

Plan for enzymatic joining of synthetic oligonucleotide segments in total synthesis of gene for precursor of tyrosine tRNA. Grouping of the total chemically synthesized segments into eight duplexes also includes the promoter and processing signal sequences derived in Figs. 7 and 8 , respectively. Reprinted with permission ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 10
Figure 10

Totally synthetic DNA duplex corresponding to entire sequence (126 nucleotides) of precursor to tyrosine-suppressor tRNA. The numbers and distances between the carets show the oligonucleotides synthesized chemically. The carets indicate the sites where joining was accomplished by the use of polynucleotide ligase. The three bold strips placed between the strands of the duplex at different positions indicate the sites where the preformed duplexes were joined to each other.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 11
Figure 11

Synthetic tyrosine-suppressor tRNA gene (from bottom left), showing terminal RI endonuclease-specific sequence. Then follow a 51-nucleotide-long promoter region, a 126-nucleotide-long DNA corresponding to the precursor RNA, and finally, a 25-nucleotide-long region. In the 25-nucleotide region, 16 nucleotides belong to the natural sequence that includes the RI endonuclease-specific sequence. Additional duplex segments (top right) are the sequences as they are naturally found in these regions; these segments continue in the two directions. Reprinted with permission ( ).

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 12
Figure 12

Cloning of synthetic gene for tyrosine-suppressor tRNA. The vector used was a derivative of bacteriophage λ with two amber mutations (A- and B-). Digestion with the RI endonuclease-specific restriction enzyme excised a portion of the phage DNA, as shown. Subsequent addition of the synthetic gene and ligase gave the circular phage, which was tested for growth by virtue of suppression of the amber mutations.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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Image of Figure 13
Figure 13

Experiment showing phage growth in following insertion of synthetic amber-suppressor gene. The two culture dishes show (A) bacteriophage λ with amber mutation used as the vector for the synthetic gene and (B) the same vector with inserted suppressor tRNA gene, prepared as in Fig. 12 . As is seen, phage plaques are present in B.

Citation: Khorana H. 1995. Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, p 5-16. In tRNA. ASM Press, Washington, DC. doi: 10.1128/9781555818333.ch2
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References

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