Ribonuclease T1
Encyclopedia
Ribonuclease T1 is a fungal
endonuclease
that cleaves single-stranded RNA
after guanine
residues, i.e., on their 3' end; the most commonly studied form of this enzyme
is the version found in the mold
Aspergillus oryzae
. Owing to its specificity for guanine, RNase T1 is often used to digest denatured RNA
prior to sequencing. Similar to other ribonucleases such as barnase
and RNase A
, ribonuclease T1 has been popular for folding studies.
Structurally, ribonuclease T1 is a small α+β protein (104 amino acid
s) with a four-stranded, antiparallel beta sheet
covering a long alpha helix
(almost five turns). RNase T1 has two disulfide bonds, Cys2-Cys10 and Cys6-Cys103, of which the latter contributes more to its folding stability; complete reduction of both disulfides usually unfolds the protein, although its folding can be rescued with high salt concentrations.
RNase T1 also has four proline
s, two of which (Pro39 and Pro55) have cis isomer
s of their X-Pro peptide bond
s. Nonnative isomers of these prolines can retard conformational folding dramatically, folding on a characteristic time scale of 7,000 seconds (almost two hours) at 10°C and pH 5.
Fungus
A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds , as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from plants, animals, and bacteria...
endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
that cleaves single-stranded RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
after guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
residues, i.e., on their 3' end; the most commonly studied form of this enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
is the version found in the mold
Mold
Molds are fungi that grow in the form of multicellular filaments called hyphae. Molds are not considered to be microbes but microscopic fungi that grow as single cells called yeasts...
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae is a filamentous fungus . It is used in Chinese and Japanese cuisine to ferment soybeans. It is also used to saccharify rice, other grains, and potatoes in the making of alcoholic beverages such as huangjiu, sake, and shōchū...
. Owing to its specificity for guanine, RNase T1 is often used to digest denatured RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
prior to sequencing. Similar to other ribonucleases such as barnase
Barnase
Barnase is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. It is synthesized and secreted by the bacterium Bacillus amyloliquefaciens, but is lethal to the cell when expressed without its inhibitor barstar...
and RNase A
Ribonuclease A
Ribonuclease A is a pancreatic ribonuclease that cleaves single-stranded RNA. Bovine pancreatic RNase A is one of the classic model systems of protein science.-History:...
, ribonuclease T1 has been popular for folding studies.
Structurally, ribonuclease T1 is a small α+β protein (104 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s) with a four-stranded, antiparallel beta sheet
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
covering a long alpha helix
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
(almost five turns). RNase T1 has two disulfide bonds, Cys2-Cys10 and Cys6-Cys103, of which the latter contributes more to its folding stability; complete reduction of both disulfides usually unfolds the protein, although its folding can be rescued with high salt concentrations.
RNase T1 also has four proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
s, two of which (Pro39 and Pro55) have cis isomer
Isomer
In chemistry, isomers are compounds with the same molecular formula but different structural formulas. Isomers do not necessarily share similar properties, unless they also have the same functional groups. There are many different classes of isomers, like stereoisomers, enantiomers, geometrical...
s of their X-Pro peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...
s. Nonnative isomers of these prolines can retard conformational folding dramatically, folding on a characteristic time scale of 7,000 seconds (almost two hours) at 10°C and pH 5.