RNase T
Living cells are filled with RNases, but not highly destructive ones like our digestive
RNase. Instead, our cellular RNases are specific and perform specialized tasks. Many of
these choreograph the timely degradation of messenger RNA strands after they have
finished their jobs. Other RNases play essential roles in the synthesis of more permanent
forms RNA, such as transfer RNA and ribosomal RNA. Many RNA molecules are built
as long precursors, which are then trimmed to the final functional length. In Escherichia
coli, RNase T has the job of performing the last cut in transfer RNA production, forming
the proper CCA 3' end that is used to carry amino acids. It also performs the final
trimming in the 5S and 23S ribosomal RNA molecules, which have a CC sequence at
their 3' ends. In other organisms, the process is not quite this simple, since some transfer
RNA molecules are not synthesized with their CCA sequence built in. In those cases,
other RNases clip the strand, and a dedicated enzyme (tRNA nucleotidyl transferase)
builds a new CCA on the exposed end.
Trimming Transfer RNA
Studies of RNase T with various substrates identified several properties that are essential
for its function. First, unlike many other RNases, the enzyme can trim RNA chains very
close to a double-stranded region, in fact, trimming overhanging ends all the way back to
form a blunt end. In its normal function, RNase T doesn't go this far, but it does get fairly
close, leaving only a few overhanging bases. Second, RNase T does not cleave CC
sequences well. This explains the specificity of ribosomal RNA processing, but still
leaves a bit of mystery about how it leaves the terminal CCA sequence in transfer RNA.
This may be due to the observation that the enzyme is not processive: it diffuses up to
the transfer RNA, clips off one base, then diffuses away. Since aminoacyl-tRNA
synthetases are so plentiful in the cell, they rapidly add an amino acid to the new CCA
end, protecting it from further digestion. If RNase T does clip off the terminal A,
however, it will definitely stop at the exposed CC, and the A can be added back on by
tRNA nucleotidyl transferase.
Structure Explaining Function
Researchers at the Midwest Center for Structural Genomics have solved the structure of
RNase T from two different bacteria as part of a collaborative effort with the University
of Miami School of Medicine (PDB entries
2f96,
shown here, and
2is3).
The structure
reveals the atomic basis of RNase T function. Overall, RNase T is a dimeric enzyme with
two identical active sites. Each active site is composed of amino acids from both chains.
A nucleotide recognition patch is donated by one chain. It is comprised of a collection of
arginine and lysine amino acids (in blue in the lower image) that are expected to interact
with the phosphate backbone. The other chain contains the catalytic machinery, which is
comprised of a collection of conserved acidic amino acids (in red) that coordinate two
magnesium ions. Four phenylalanine amino acids (in white), one from one chain and
three from the other, are thought to provide the specificity that disfavors transfer RNA
sequences with cytosine nucleotides. In other cases of RNA-protein interaction,
phenylalanine shows weaker interaction with cytosine bases than with other bases.
Click on the image for an interactive Jmol view.
References
Y. Zuo, H. Zheng, Y. Wang, M. Chruszcz, M. Cymborowski, T. Skarina, A. Savchenko,
A. Malhotra and W. Minor (2007) Crystal structure of Rnase T, and exoribonuclease
involved in tRNA maturation and end turnover. Structure 15, 417-428.
Y. Zuo and M. P. Deutscher (2002) The physiological role of Rnase T can be explained
by its unusual substrate specificity. Journal of Biological Chemistry 277, 29654-29661.