rRNA endonuclease (, ''alpha-sarcin'') is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

that catalyses the

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

of the phosphodiester linkage between

guanosine Guanosine (symbol G or Guo) is a purine nucleoside comprising guanine attached to a ribose ( ribofuranose) ring via a β-N9- glycosidic bond. Guanosine can be phosphorylated to become guanosine monophosphate (GMP), cyclic guanosine monophosp ...

and

adenosine Adenosine (symbol A) is an organic compound that occurs widely in nature in the form of diverse derivatives. The molecule consists of an adenine attached to a ribose via a β-N9- glycosidic bond. Adenosine is one of the four nucleoside build ...

residues at one specific position in the

28S rRNA 28S ribosomal RNA is the structural ribosomal RNA (rRNA) for the large subunit (LSU) of eukaryotic cytoplasmic ribosomes, and thus one of the basic components of all eukaryotic cells. It has a size of 25S in plants and 28S in mammals, hence th ...

rat Rats are various medium-sized, long-tailed rodents. Species of rats are found throughout the order Rodentia, but stereotypical rats are found in the genus ''Rattus''. Other rat genera include '' Neotoma'' (pack rats), '' Bandicota'' (bandicoo ...

ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...

s. This ribotoxin also acts on

bacterial Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among the ...

rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...

. A

ribosome-inactivating protein A ribosome-inactivating protein (RIP) is a protein synthesis inhibitor that acts at the eukaryote, eukaryotic ribosome. This protein family describes a large family of such proteins that work by acting as rRNA N-glycosylase (EC 3.2.2.22). They ina ...

produced by the mold '' Aspergillus giganteus'', alpha-sarcin cleaves the portion of ribosomal RNA that forms the small ribosomal substrate. The high specificity of alpha-sarcin and its efficiency of cleavage are point of study and also account for this protein's very high toxicity level.

Structure

It is believed that the

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a conditionally essential amino acid with a polar side group. The word "tyrosine" is ...

amino acid found along the amino acid sequence of alpha-sarcin allows for the specificity when alpha-sarcin binds to the rRNA. It is the alcohol group found on the tyrosine amino acid that allows for this binding. This was determined in tests that removed the alcohol group, replacing tyrosine with

phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the chemical formula, formula . It can be viewed as a benzyl group substituent, substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of ...

, and the binding affinity was greatly reduced. The region of the DNA that makes alpha-sarcin is highly conserved, along with the corresponding sequence on the targeted ribosome. The corresponding sequence on the targeted ribosome is a centered around a

guanine Guanine () (symbol G or Gua) is one of the four main nucleotide bases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine ( uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside ...

nucleotide located on what is called the "bulged-G motif".

Specificity

Alpha-sarcin is remarkable in its cleavage specificity. It interacts with a single bond in the targeted ribosome and breaks it, causing the ribosome to be inactivated. The bond in question is the phosphodiester bond within the sarcin/ricin loop (SRL) of the rRNA. The SRL region of the RNA was named after the alpha-sarcin toxin that targets it. The targeted bond is located within the GAGA tetraloop of the RNA in between a guanine and adenine nucleotide. Other ribotoxins also cleave the RNA of the ribosomes, however there are many more points of cleavage- indicating much less specificity. The specificity of alpha-sarcin is so high, that alpha-sarcin can recognize the SRL segment of the ribosome without the rest of the ribosome present. SRLs fold independently, creating the same structure as when they are in a ribosome. This reaffirms the idea that it is the affinity of alpha-sarcin for this specific region of the ribosome that causes the two to bind and react. The key recognition nucleotide on the ribosome is a

nucleotide located six nucleotides upstream from the cleave site (this is the same as the above-mentioned "g-bulge" region).

Conditions for reaction

The conditions that allow for the recognition and cleavage include the salinity of the environment. With increased salt concentration, there is increased competition for the alpha-sarcin to reach the "G-bulge". This is due to the electrostatic interactions between the cationic side chains of the amino acids of the alpha-sarcin and the phosphates of the ribosome chain. More salt interferes with these two interactions. The overall rate constant for the SRL cleavage reaction is second order (k2/K1/2 of 108M-1s-1). This means the reaction rate is directly proportional to the concentrations of the reactant squared. The rate does not appear to be dependent on physical steps, i.e. the two molecules being able to locate each other in solution is not a factor in how quickly they react. This was determined by observing the rate of the reaction under varying viscosities. The dissociation of the products, the separation of the two molecules, also has no effect on the rate. It is suggested that the rate determining step for SRL cleaves occurs within the chemical cleavage of the phosphodiester bond. Once the alpha-sarcin have cleaved the ribosome, the resulting pieces, P1 and P2 are cleaved further, with a particular affinity towards A and G sites. However, this later cleaving has a much lower rate of reaction.4 This further supports the notion that alpha-sarcin depends on the folded structure of the RNA for recognition and cleavage, but not necessarily the rest of the molecule. The cleavage rate for an unfolded ssRNA containing the GA sequence is three times less than the full and folded SRL sequence.

References

External links

* {{Portal bar, Biology, border=no Enzymes Ribosome-inactivating proteins EC 3.1.27