1887

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

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in
Zoomout

Transfer RNA: Discovery, Early Work, and Total Synthesis of a tRNA Gene, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555818333/9781555810733_Chap02-2.gif

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

Key Concept Ranking

DNA Polymerase I
0.52884614
DNA Synthesis
0.4699142
Integral Membrane Proteins
0.43450728
0.52884614
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint
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
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555818333.chap2
1. Altman, S.,, and J. Smith. 1971. Tyrosine tRNA precursor molecule polynucleotide sequence. Nature New Biol. 233:35.
1a. Asenjo, A. B.,, J. Rim,, and D. D. Oprian. Molecular determinants of human red/green color discrimination. Neuron, in press.
2. Berg, P. 1961. Specificity in protein synthesis. Annu. Rev. Biochem. 30:293324.
3. Besmer, P.,, R. C. Miller, Jr.,, M. H. Caruthers,, A. Kumar,, K. Minamoto,, J. H. van de Sande,, N. Sidarova,, and H. G. Khorana. 1972. Studies on polynucleotides. CXVII. Hybridization of polydeoxynucleondes with tyrosine transfer RNA sequences to the r-strand of d80psu+III. J. Mol. Biol. 72:503522.
4. Caruthers, M. H. 1991. Chemical synthesis of DNA and DNA analogues. Acc. Chem. Res. 24:278284.
5. Caruthers, M. H.,, K. Kleppe,, J. H. van de Sande,, V. Sgaramella,, K. L. Agarwal,, H. Buchi,, N. K. Gupta,, A. Kumar,, E. Ohtsuka,, U. L. RajBhandary,, T. Terao,, H. Weber,, T. Yamada,, and H. G. Khorana. 1972. Studies on polynucleotides. CXV. Total synthesis of the structural gene for an alanine transfer RNA from yeast (13). Enzymatic joining to form the total DNA duplex./. Mol. Biol. 72:475492.
6. Crick, F. H. C. 1966. The genetic code—yesterday, today, and tomorrow. Cold Spring Harbor Symp. Quant. Biol. 31:39.
7. Crick, F. H. C. 1966. Codon-anticodon pairing: the wobble hypothesis.J. Mol. Biol. 19:548555.
8. Dunn, R. J.,, R. Belagaje,, E. L. Brown,, and H. G. Khorana. 1981. The synthesis and cloning of two tyrosine suppressor tRNA genes with altered promoter sequences. J. Biol. Chem. 256:61096118.
9. Eriani, G.,, M. Delarue,, O. Poch,, J. Gangloff,, and D. Moras. 1990. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature (London) 347:203206.
9a. Gilbert, W.,, and A. Maxam. 1973. The nucleotide sequence of the lac operator. Proc. Natl. Acad. Sci. USA 70:35813584.
10. Gillam, I.,, D. Blew,, R. C. Warrington,, M. von Tigerstrom,, and G. M. Tener. 1968. A general procedure for the isolation of specific transfer ribonucleic acids. Biochemistry 7:34593468.
11. Gillam, I.,, S. Millward,, D. Blew,, M. von Tigerstrom,, E. Wimmer,, and G. M. Tener. 1967. The separation of soluble ribonucleic acids on benzoylated diethylaminoethylcellulose. Biochemistry 6:30433056.
12. Goodman, H. M.,, J. N. Abelson,, A. Landy,, S. Brenner,, and J. D. Smith. 1968. Amber suppression: a nucleotide change in the anticodon of a tyrosine transfer RNA. Nature (London) 217:10191024.
13. Gupta, N. K.,, E. Ohtsuka,, H. Weber,, S. H. Chang,, and H. G. Khorana. 1968. Studies on polynucleotides. LXXXVII. The joining of short deoxyribopolynucleotides by DNA-joining enzymes. Proc. Natl. Acad. Sci. USA 60:285292.
14. Heckman, J. E.,, J. Sarnoff,, B. Alzner-DeWeerd,, S. Yin,, and U. L. RajBhandary. 1980. Novel features in the generic code and codon reading patterns in Neurospora crassa mitochondria based on sequence on six mitochondrial tRNA's. Proc. Natl. Acad. Sci. USA 77:31593163.
15. Hoagland, M. R.,, E. B. Keller,, and P. C. Zamecnik. 1956. Enzymatic carboxyl activation of amino acids. J. Biol. Chem. 218:345358.
16. Hoagland, M. R.,, M. L. Stephenson,, J. F. Scott,, I. H. Hecht,, and P. C. 1958. A soluble ribonucleic acid intermediate in protein synthesis. J. Biol. Chem. 231:241257.
17. Holley, R. W. 1957. An alanine-dependent, ribonuclease- inhibited conversion of AMP to ATP, and its possible relationship to protein synthesis. J. Am. Chem. Soc. 79:658662.
18. Holley, R. W.,, J. Apgar,, G. A. Everett,, J. T. Madison,, M. Marquisee,, S. H. Merrill,, J. R. Penswick,, and A. Zamir. 1965. Structure of a ribonucleic acid. Science 147:14621465.
19. Khorana, H. G. 1960. Synthesis of nucleotides, nucleotide coenzymes and polynucleotides. Fed. Proc. 19:931941.
20. Khorana, H. G. 1968. Synthesis in the study of nucleic acids, p. 17. Proceedings of the Seventh International Congress of Biochemistry. Tokyo.
21. Khorana, H. G. 1968. Synthesis in the study of nucleic acids. Biochem. J. 109:709725.
22. Khorana, H. G. 1968. Nucleic acid synthesis. Pure Appl. Chem. 17:349381.
22a. Khorana, H. G. 1978. Studies on nucleic acids: total synthesis of a biologically functional gene. Bioorg. Chem. 7:351393.
23. Khorana, H. G. 1992. Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J. Biol. Chem. 267:14.
24. Khorana, H. G. 1993. Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin. Proc. Natl. Acad. Sci. USA 90:11661171.
25. Khorana, H. G.,, K. L. Agarwal,, H. Buchi,, M. H. Caruthers,, N. Gupta,, K. Kleppe,, A. Kumar,, E. Ohtuska,, U. L. RajBhandary,, J. H. van de Sande,, V. Sgaramella,, T. Terao,, H. Weber,, and T. Yamada. 1972. Studies on polynucleotides. CHI. The total synthesis of the structural gene for alanine transfer ribonucleic acid from yeast (general introduction). J. Mol. Biol. 72:209217.
26. Kim, S. H.,, F. L. Suddath,, G. J. Quigley,, A. McPherson,, J. L. Sussman,, A. H. J. Wang,, N. C. Seeman,, and A. Rich. 1974. Three-dimensional tertiary structure of yeast phenylalanine transfer RNA. Science 185:435440.
27. Kleppe, K.,, E. Ohtsuka,, R. Kleppe,, I. J. Molineux,, and H. G. Khorana. 1971. Studies on polynucleotides. XCVI. Repair replication of short synthetic DNAs as catalyzed by DNA polymerases.J. Mol. Biol. 56:341361.
28. Kleppe, R.,, T. Sekiya,, P. C. Loewen,, K. Kleppe,, K. L. Argarwal,, M. Fridkin,, E. Jay,, A. Kumar,, R. C. Miller,, K. Minamoto,, A. Panet,, U. L. RajBhandary,, B. Ramamoorthy,, N. Sidorova,, T. Takei,, K. H. van de Sande,, and H. G. Khorana. 1976. Studies on polynucleotides. CXLI. Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from E. coli (11). Enzymatic joining to form the total DNA duplex. J. Biol. Chem. 251:667675.
29. Krebs, M. P.,, E. N. Spudich,, H. G. Khorana,, and J. L. Spudich. 1993. Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium. Proc. Natl. Acad. Sci. USA 90:34863490.
30. Madison, J. T.,, G. A. Everett,, and H. K. Kung. 1966. On the nucleotide sequence of yeast tyrosine transfer RNA. Cold Spring Harbor Symp. Quant. Biol. 31:409416.
31. Normanly, J.,, L. G. Kleina,, J.-M. Masson,, J. Abelson,, and J. H. Miller. 1990. Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. J. Mol. Biol. 213:719726.
32. Oprian, D. D.,, A. B. Asenjo,, N. Lee,, and S. L. Pelletier. 1991. Design, chemical synthesis, and expression of genes for the three human color vision pigments. Biochemistry 30:1136711372.
33. Peterson, E. A.,, and H. A. Sober. 1956. Chromatography of proteins. I. Cellulose ion-exchange adsorbents. J. Am. Chem. Soc. 78:751755.
34. RajBhandary, U. L.,, S. H. Chang,, H. J. Gross,, F. Harada,, F. Kimura,, and S. Nishimura. 1969. E. coli tyrosine transfer-RNA— primary sequence and direct evidence for base-pairing between the terminal sequences. Fed. Proc. 28:409.
35. RajBhandary, U. L.,, A. Stuart,, R. D. Faulkner,, S. H. Chang,, and H. G. Khorana. 1966. Nucleotide sequence studies on yeast phenylalanine sRNA. Cold Spring Harbor Symp. Quant. Biol. 31:425434.
36. Razzell, W. E.,, and H. G. Khorana. 1958. The stepwise degradation of thymidine oligonucleotides by snake venom and spleen phosphodiesterase. J. Am. Chem. Soc. 80:1770.
37. Robertus, J. D.,, J. E. Ladner,, J. T. Finch,, D. Rhodes,, R. S. Brown,, B. F. C. Clark,, and A. Mug. 1974. Structure of yeast phenylalanine tRNA at 3 X resolution. Nature (London) 250:546551.
38. Ryan, M. J.,, E. L. Brown,, T. Sekiya,, H. Kupper,, and H. G. Khorana. 1979. Total synthesis of a tyrosine suppressor transfer RNA gene (18). In vitro and in vivo expression of the cloned chemically synthesized tyrosine suppressor transfer RNA gene. J. Biol. Chem. 254:58175826.
38a. Saks, M. E.,, J. R. Sampson,, and J. N. Abelson. 1994. Science 263:191197.
39. Sanger, E.,, G. G. Brownlee,, and B. G. Barrell. 1965. A two-dimensional fractionation procedure for radioactive nucleotides. J. Mol. Biol. 13:373398.
39a. Schimmel, P.,, R. Giege,, D. Moras,, and S. Yokayama. 1993. Proc. Natl. Acad. Sci. USA 90:87638768.
40. Sekiya, T.,, R. Contreras,, H. Kupper,, A. Landy,, and H. G. Khorana. 1976. Escherichia coli tyrosine transfer ribonucleic acid genes: nucleotide sequence of their promoters and of the regions adjoining the C-C-A ends. J. Biol. Chem. 251:51245140.
41. Sekiya, X.,, R. Contreras,, T. Takeya,, and H. G. Khorana. 1979. Total synthesis of a tyrosine suppressor transfer RNA gene (17). Transcription in vitro of the synthetic gene and processing of the primary transcript to transfer RNA. J. Biol. Chem. 254:58025816.
42. Sekiya, T.,, T. Takeya,, E. L. Brown,, R. Belagaje,, R. Contreras,, H. J. Fritz,, M. J. Gait,, R. G. Lees,, M. J. Ryan,, H. G. Khorana,, and K. E. Norris. 1979. Total synthesis of a tyrosine suppressor transfer RNA gene (16). Enzymatic joinings to form the total 207 base-pair long DNA. J. Biol. Chem. 254:57855801.
43. Sekiya, T.,, T. Takeya,, R. Contreras,, H. Kupper,, H. G. Khorana,, and A. Landy,. 1976. Nucleotide sequences at the two ends of the E. coli tyrosine tRNA genes and studies on the promoter, p. 455472. In R. Losick, and M. Chamberlin (ed.), RNA Polymerase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
44. Sekiya, X.,, H. van Ormondt,, and H. G. Khorana. 1975. Studies on polynucleotides. CXXVIII. The nucleotide sequence in the promoter region of the gene for an E. coli transfer ribonucleic acid. J. Biol. Chem. 250:10871098.
45. Soli, D.,, and U. L. RajBhandary. 1967. Studies on polynucleotides. LXXVI. Specificity of tRNA for codon recognition as studied by amino acid incorporation. J. Mol. Biol. 29:113124.
46. Tomlinson, R. V.,, and G. M. Tener. 1963. The effect of urea, formamide and glycols on the secondary binding forces in the ion-exchange chromatography of polynucleotides on DEAE-cellulose. Biochemistry 2:697702.
46a. Wu, R.,, and E. Taylor. 1971. Nucleotide sequence analysis of DNA. II. Complete nucleotide sequences of the cohesive ends of bacteriophage X DNA.J. Mol. Biol. 57:491511.
47. Zachau, H. G.,, G. Acs,, and F. Lipmann. 1958. Isolation of adenosine amino acid esters from a ribonuclease digest of soluble, liver ribonucleic acid. Proc. Natl. Acad. Sci. USA 44:885889.
48. Zachau, H. G.,, D. Dutting,, H. Feldmann,, F. Melchers,, and W. Karau. 1966. Serine specific transfer ribonucleic acids. XIV. Comparison of nucleotide sequences and secondary structure models. Cold Spring Harbor Symp. Quant. Biol. 31:417424.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error