Structural Biochemistry/Nucleic Acid/RNA/RNA modification/Ribonucleases

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Ribonuclease[edit]

Ribonuclease is a type of enzyme that is capable of cleaving the phosphodiester bond between each unit of nucleic acid that form the RNA backbone. A phosphodiester bond in a single RNA strand is formed by the linkage between the 3’ carbon atom of one ribose sugar molecule and the 5’ carbon atom of another ribose sugar attached to an adjacent nucleoside. These enzymes have overlapping functions as the small nuclear RNAs acting on mRNAs, such that they catalyze RNA degradation by breaking down RNA into shorter partial strands. Ribonuclease can be grouped into two categories: endoribonucleases and exoribonucleases.

300px a picture of two RNA nucleotides with base adenine and cytosine

Endoribonucleases[edit]

Endoribonulcases, much like the restriction endonucleases, are able to recognize certain amino acid groups in RNA and cleave the RNA there. These enzymes are able to break apart both single or double-stranded RNAs.

Exoribonucleases[edit]

Exoribonucleases cleave RNA by removing terminal nucleotides from either the 5’ end or the 3’ end of the RNA strand. Enzymes that remove nucleotides from the 5' end are called 5'-3' exoribonucleases, and enzymes that remove nucleotides from the 3' end are called 3'-5' exoribonucleases.


Both endonucleases and exoribonucleases can be further broken down into sub-classes of ribonucleases based on their chemical cleaving mechanisms, such as phosphorolytic and hydrolytic activations. They exist in all kingdoms of life, the bacteria, archaea, and eukaryotes. They are involved in the degradation of many different RNA species, such as messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.

The left side shows the hydrolytic activity of RNases in which a water molecule intercepts the 3’ ester bond between the phosphate group and the 5’ –OH group of the adjacent sugar, breaking off one nucleotide. The left side shows the phosphorolytic activity of RNases in which a phosphate group intercepts the 3’ ester bond between the phosphate group and the 5’ –OH group of the adjacent sugar, breaking off a nucleotide with two phosphate groups on it. The left side shows the hydrolytic activity of RNases in which a water molecule intercepts the 3’ ester bond between the phosphate group and the 5’ –OH group of the adjacent sugar, breaking off one nucleotide. The left side shows the phosphorolytic activity of RNases in which a phosphate group intercepts the 3’ ester bond between the phosphate group and the 5’ –OH group of the adjacent sugar, breaking off a nucleotide with two phosphate groups on it.

500px


The left side shows a strand of mRNA, protected by a G-cap and a poly-A-tail. The Dcp1/2 protein recognizes the G-cap at the 5’ end of the RNA, takes it away, and exoribonuclease Xrn1 comes and starts chewing off the RNA strand from the 5’ end. The right side shows the deadenylase protein recognizing the poly-A-tail and starts chewing it off. Then comes the exosome, another exoribonuclease that degrade RNA from the 3’ to 5’ direction. The middle shows an endoribonuclease binding to a specific sequence within the RNA and cleaving it internally.

Important RNase[edit]

RNase A[edit]

The structure of RNase A was first crystalized 50 years ago. It was the first enzyme and the third protein whose amino sequence was determined. It is a single chain protein that contains 4 disulfide bridges. It contains 124 amino residues and 19 out of the 20 amino acids, except for tryptophan. These enzymes were found in the exocrine cells of the bovine pancreas. They are very tough and highly stabilized enzymes, mostly due to their structures and folding patterns. Its molecular formula is C575H907N171O192S12 and the general structure consists of 2 sheets of alpha helices and beta sheets cross-linked by four disulfide bridges. RNase A has basic properties. It specifically attacks at the 3’ phosphate of a pyridine nucleotide. The cleavage involves simply two steps: 1). the 3’,5’-phosphodiester bond is cleaved to generate a 2’,3’-cyclic phosphodiester intermediate and 2). the cyclic phosphodiester is hydrolyzed to a 3’-monophosphate.

Example: pG-pG-pC-pA-pG undergoes RNase A cleavage would result in 2 sequences: pG-pG-pCp and A-pG.

RNase A can be activated by potassium and sodium salt and inhibited by alkylation of His12 or His119 in initiate RNA cleaving. It utilizes both phosphorolytic and hydrolytic activities to cleave a strand of RNA.

RNase P[edit]

RNase P.png

Sidney Altman discovered and named RNase P while working in the Laboratory of Molecular Biology in Cambridge, England, focusing on the functions of tRNA. He proposed that by altering spatial relationships in tRNA, either by insertion of new nucleotides or deletion of existing nucleotides, would affect or change the function of the tRNA. His experiments with E. coli. have shown that mutated tRNAs could not develop into a full mature tRNA, which in turn could not serve its functions of delivering amino acids during protein synthesis. However, these dysfunctional tRNAs quickly resolved back to wild-type tRNAs. By isolating the DNA to RNA transcript of tRNA, Altman had found that there are extra nucleotides hanging off of the 5’ and 3’ ends of tRNAs. When these tRNAs were introduced to a live medium, an enzyme was observed to cut off the extra nucleotides through the cleave of a phosphodiester bond, exposing the 5’ end of the molecule. This RNase was different than other previous known RNase because of its specificity at the 5’ end of the tRNA. Altman also showed that RNase P-like activities were present in cells extracted from a variety of organisms, including humans. RNase P is unique that it is ribozyme. While it cleave other RNAs, it cleaves itself as well, meaning it self-destructs during reaction. It is a single stranded protein containing 120 amino residues. They are found in many organisms such as archaea, bacteria and eukarya as well as chloroplasts in plants. The make-up of RNase P differ from one organism to another, but their functionalities are the same because of orthogonal properties. RNase P is a crucial component in the production of functional tRNA molecules.

RNase T2[edit]

Screen Shot 2012-11-19 at 11.29.03 PM.png

T2 family Ribonucleases are considered to be transferase type RNases and are distinguished from the RNaseA and RNaseT1 protein families based on three features.

- First of all T2 ribonucleases are more evenly distributed and are found in bacteria, plants, protozoans, animals and even viruses, whereas RNase T1 enzymes exist only in bacterial and fungal organisms and RNaseA family enzymes are highly represented in animals.

- Secondly, the optimal pH of activity of many T2 ribonucleases is between 4 and 5. By contrast, RNaseT1 and RNaseA families have optimal pH activity at alkaline (pH 7-8) or weakly acidic (pH ~7).

- Thirdly, T2 ribonucleases do not discriminate their cleavage sites. T2 families generally cleave at all four bases, whereas RNaseA and RNaseT1 families tend to be specific for pyrimidine or guanosiine bases respectively.

The biological role for T2 Ribonucleases varies. These endoribonucleases are ubiquitously represented in organisms across kingdoms and have been show to perform a variety of functions in different organisms besides it's ability to hydrolyze RNA. Some examples of biological roles include the scavenging of nucleic acids, the degradation of self-RNA, modulation of a host immune response, and serving as cellular cytotoxins.

Other T2 RNase Properties[edit]

T2 RNase are transferase-type RNases and catalyze the cleavage of ssRNA (single-stranded RNA) through a 2',3'-cyclic phosphate intermediate. The result of this catalyzed reaction are mono- or oligonucleotides with a 3' phosphate group. Typically, these RNases are secreted from the cell or specific special locations within the cell such as vacuoles, which may prove important to how their activity is modulated within the cell. This family of RNases has a specific structure and mechanism that is well known from x-ray crystallography. Likewise, crystallography has defined specific places such as specific binding sites, called B1 for sites with a 5' end and B2 for site with a 3' end, as well as a core made up of alpha and beta structures. Additionally, the catalysis of T2 RNase starts with one to three histidine residues. It should be noted that alteration or mutation of these residues causes inactivation of the RNase. The two main steps of this catalysis are transphosphorylation and hydrolysis. Further study is being conducted in the following areas for these specific RNases:

  1. Discovering how RNases from this family can function independent of catalysis
  2. Mutational anaylsis to determine the regions necessary for nuclease-independent functions
  3. How these RNases enter the cell to reveal how proteins cross membranes, while many things cannot

References[edit]

1. T2 Family ribonucleases: ancient enzymes with diverse roles. Luhtala N, Parker R. Trends Biochem Sci. 2010

2. Raines, Ronald T. "Ribonuclease A." Chem Rev. Wisconsins: University of Wisconsins, 1998. 1045-065. Print.

3. Gopalan, Venkat, Agustin Vioque, and Sidney Altman. "RNase P: Variations and Uses*." RNase P: Variations and Uses. JBC Papers in Press, 10 Dec. 2011. Web. 20 Nov. 2012. <http://www.jbc.org/content/277/9/6759.full>.

4. Cuchillo, C. M.; Nogués, M. V.; Raines, R. T. (2011). "Bovine pancreatic ribonuclease: Fifty years of the first enzymatic reaction mechanism". Biochemistry 50: 7835-7841. PMC 3172371. PMID 21838247.

5. J. Holzmann, P. Frank, E. Löffler, K. Bennett, C. Gerner & W. Rossmanith (2008). "RNase P without RNA: Identification and functional reconstitution of the human mitochondrial tRNA processing enzyme". Cell 135 (3): 462–474. doi:10.1016/j.cell.2008.09.013. PMID 18984158.