Structural Basis For Dual-Substrates Methyltransferease SpRlmCD

Translation is one of the most important processes in genetics,and ribosome is the molecular machine responsible for protein translation.Modifications of rRNAs are required in ribosome maturation process.The modifications of rRNAs affects the structure and function of ribosome,and are closely related to the binding of antibiotics.In the past decades,the apo-form/complex structure of enzymes responsible for rRNA modification has been resolved.These structures reveal substrate specificity and enzyme mechanism.Most of the rRNA methyltransferases in bacteria(eg.E.coli)are responsible for the modification of specific site or two adjacent sites.In E.coli,there are two 23S rRNA m5U modifications:m5U1939 and m5U747.The structure of E.coli RlmD,which is responsible for m5U1939 modification,has been resolved.However,the structure of E.coli RlmC,which is responsible for m5U747 modification,has not been resolved.Recently,it was found that Streptococcus pneumoniae RlmCD are responsible for both m5U modifications.We solved the crystal structures of Streptococcus pneumoniae methyltransferase RlmCD complexes:RlmCD-SAH-U747RNA ternary complex and RlmCD-SAH-U1939RNA ternary complex.Through the structures of ternary complexes,we found that RlmCD can specifically recognize U747 RNA and U1939 RNA.Compared to ec RlmD,spRlmCD has two additional loops.These two loops and L344 residue let RlmCD binding U1939 RNA in a different style than RlmD.These two loops and F145,F146,H151 residues specifically recognize U747 RNA.Moreover,we detected the enzyme activities of wild-type and mutant-types by liquid scintillation counting,we found that the mutation of RlmCD greatly reduced the enzyme activity efficiency.Through the crystal structures and invtro MTase assay,we revealed that RlmCD has a unique structure responsible for dual-sites methyltransferase activity.The additional loops and F146,H151 and Q162 residues are the structural basis for dual-site methyltransferase activity.Furthermore,these residues are conservative in Bacillus subtilis RlmCD.The second part of this paper is the single molecule FRET experiment of H2A.Bbd nucleosome.The nucleosome core particle(NCP)is the fundamental repeating unit in chromatin.H2A,H2B,H3 and H4 form histone octamer,and DNA is wrapped around the octamer to form NCP.H2A.Bbd is the most unconservative histone variants,and it plays an important role in spermatogenesis.H2A.Bbd NCP binds the DNA ends less tightly,and have a loose structure.Chaperone NapI is responsible for H2A.Bbd-H2B dimer assemble and disassemble in the nucleosomes.The interaction or conformational changes between biomolecules often involve nanoscale(1-10 nm)distance changes.Single molecule fluorescence resonance energy transfer(smFRET)is a spectroscopic technique that can observe distance changes at the nanoscale sensitively.In this paper,we successfully assembled the H2A.Bbd NCP and observed the stability at different temperatures.We introduced cy3 and cy5 fluorophores into the DNA of H2A.Bbd NCP.FRET of H2A-NCP and FRET of H2A.Bbd-NCP were observed by TIRF microscopy.FRET efficiency in H2A.Bbd-NCP is lower,indicating that the distance between the ends of the DNA is farther,and also suggesting that H2A.Bbd NCP binds the DNA ends less tightly.