The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

Abstract

Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 59 end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 39 ends of most guide RNAs have~15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G d U wobble base pairs. To gain insights into the structure of the U-helix, we crystallized 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 59-AGA-39/59-UUU-39 base-pair triple. The fusion of two identical U-helices head-to-head promoted crystallization. We obtained X-ray diffraction data with a resolution limit of 1.37 Å . The U-helix had low and high twist angles before and after each G d U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins.

Interaction of Na+, K+, Mg2+ and Ca2+ counter cations with RNA

“…The same Na + ions are coordinated to О2' and О4' oxygen atoms of ribose and/or a center from a nucleobases. In the reported structures the measured Na + -O2' distances range between 255 pm and 288 pm 35,59 . Na + -О4' distances of 257 pm and 305 pm are reported 35,59 .…”

“…In the reported structures the measured Na + -O2' distances range between 255 pm and 288 pm 35,59 . Na + -О4' distances of 257 pm and 305 pm are reported 35,59 . Sodium can stabilize quadruple-stranded structures ( Figure 2H), where the Na + ion is directly bonded to four carbonyl oxygen atoms, О4U, from four uracil nucleobases (PDB 1J8G with resolution of 61 pm).…”

“…High resolution is difficult to be achieved in larger amorphous systems, like the whole ribosomal subunits 11 . Thus, the chosen structures are either parts of larger biomolecules, or short synthetic sequences [34][35][36][50][51][52][53][54][55][56][57][58][59][60] . Molecules classified as small RNAs have not been included 61 .…”

“…Respective structure sources: PDB 1J8G 52 , 397D 53 and 2ZY6 50 ; G, H, Iwith Na + ions. Respective structure sources: PDB 1L2X 35 , 1J8G 52 and 3ND3 59 ; J, K, Lwith K + ions. Respective structure sources: PDB 2OIY (J and K) 34 and 2QEK 60 .…”

“…RNA heptamer double helix (Escherichia coli transfer RNA fragment) 56 ; modified transfer RNA fragments from Escherichia coli 57 and 16-mer double-stranded RNA fragments (precursor messenger RNA from Trypanosoma brucei) 59 . The monovalent ions have affinity mainly to water molecules and RNA nucleobases, as presented in Tables 3 and 4.…”