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

Cloverleaf secondary structure model for yeast alanine-tRNA, originally proposed by Holley in 1965 ( 18 ).

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 25 with permission.

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. 32P represents the 5′-phosphate end group. Reprinted from reference 25 with permission.

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 25 with permission.

Primary nucleotide sequence of an E. coli 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 Nature New Biology [ 1 ]).

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 E. coli 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 ( 22a ).

(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 EcoRI restriction enzyme. The 10 segments (PI to P10) to be synthesized are indicated by horizontal brackets. Reprinted with permission ( 22a ).

Nucleotide sequence in double-stranded form. Note palindromic symmetry downstream from C-C-A end of tyrosine-suppressor tRNA. Reprinted with permission ( 22a ).

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 ( 22a ).

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.

Synthetic tyrosine-suppressor tRNA gene (from bottom left), showing terminal EcoRI 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 EcoRI 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 ( 22a ).

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 EcoRI 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.

Experiment showing phage growth in E. coli 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.