A Study of a Substrate Specificity of a Cytotoxin, Ricin A-Chain and an Alteration of it's Functional Domain
| Project/Area Number |
63480492
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
物質生物化学
|
|---|
| Research Institution | 山梨医科大学 |
|---|
Principal Investigator |
ENDO Yaeta Yamanashi Medical, College Biochemistry, Associate Professor, 医学部・生化学第二教室, 助教授 (40093843)
|
|---|
| Project Period (FY) |
1988 – 1990
|
| Project Status |
Completed (Fiscal Year 1990)
|
| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1990: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1988: ¥5,300,000 (Direct Cost: ¥5,300,000)
|
|---|
| Keywords | Cytotoxin / Ribosome / Ricin A-chain / alpha-Sarcin / Ribosomal RNA / Toxin domain / RNA oligomer / RNANーグリコシダ-ゼ / RNA N-グリコシダ-ゼ / RNANーグリコシダーゼ / RNAオリゴマー |
|---|
| Research Abstract |
1. RNA-Protein Interaction
a-sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 SrRNA. We have examined the requirements for the recognition by a-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vivo from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position corresponds to 4325. There is an absolute requirement for a helical stem ; however, it can be reduced from the 7 base pairs in the natural structural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition ; whereas, the bulged nucleotide does not contribute to identific
… More
ation. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG (sarcin) A leads to loss of recognition by the toxin. We proposed that the a-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacy-tRNA and of translocation ; that translocation is driven by transition in the structure of the a-sarcin domain RNA initiated by the binding of the factors, or the hydorolysis of GTP, or both ; and the toxin domain inactivates the ribosomes by preventing this transition.
2. Ribosomal RNA Identity Elements for Ricin A-Chain Recognition and Catalysis
Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose at position A-4324 in eukaryotic 28 SrRNA. The requirement for the recognition by ricin A-chain of this nucleotide and for the catalysis of cleavage were examined using a synthetic oligoribonucleotide that reproduces the sequence and the secondary structure of the RNA domain. The wild type RNA (35-mer) and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild type oligoribonucleotide the ricin A-chain catalyzed reaction has a Km of 13.55 muM and a Kcat of 0.023/min. Recognition and catalysis by ricin A-chain has an absolute requirement for an A at the position that corresponds to 4324. The helical stem is also essential, however, the number of base pairs can be reduced from the 7 found in 28 SrRNA to 3 without loss of identity. The nature of these pairs can affect catalysis : A change of the second set from one canonical pair (GC) to another (UA) reduces sensitivity to ricin A-chain ; whereas, a change of the third pair (UA to GC) produces supersensitivity. The bulged nucleotide does not contribute to identification. Hydrolysis is affected by altering the nucleotides in the universal sequence surrounding A-4324 or by changing the position in the loop of the teranucleotide GA (ricin) GA-all of these mutants have a null phenotype. If ribosomes are treated first with a-sarcin to cleave the phosphodiester bond at G-4325 ricin can still catalyze depurination at A-4324. This implies that cleavage by a-sarcin at the center of what has been presumed to be a 17 nucleotide single-stranded loop in 28 SrRNA does not produce ends that are free but rather that they are constrained. The results suggested that the loop has a complex structure and this bears on the function in protein synthesis of the a-saricin/ricin domain. Less
|
Report
(4 results)
Research Products
(34 results)
