1-Methylguanosine in Place of Y Base at Position 37 in Phenylalanine tRNA Is Responsible for Its Shiftiness in Retroviral Ribosomal Frameshifting

Abstract

Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.

tRNA biology charges to the front

“…Frameshifting was most pronounced when the last codon was UUU rather than UUC, and was progressively reduced as the Wye base was increasingly formed, since frameshifting was twofold higher than wild type in a tyw1 mutant, which has tRNA Phe with m 1 G 37 , and 1.5-fold higher than wild type in a tyw2 mutant, in which the Wye base is in an intermediate state in which only the imidazole ring is attached. This finding provides a highly satisfactory explanation of the value, and perhaps the evolution, of increased modification in the Wye base, and is consistent with previous analysis of frameshifting in vitro (Carlson et al 2001). These results nicely complement earlier work by Bjork and colleagues (Urbonavicius et al 2001(Urbonavicius et al , 2003 demonstrating that several modifications in the anticodon loop of tRNAs in E. coli, Salmonella typhimurium, or S. cerevisiae affect +1 frameshifting, but not À1 frameshifting.…”

Transfer RNA modifications that alter +1 frameshifting in general fail to affect −1 frameshifting

“…Similarly, the absence of Q34 in tRNAAsn did not influence the level of −1 frameshifting at U-UUA-AAC/U sites in the cos-cells and in vitro using the rabbit reticulocyte lysate (RRL) system (Marczinke et al 2000). Using the same in vitro system, the level of −1 frameshifting at the A-AAA-AAC site only increased 1.5-fold when the Q-deficient tRNA Asn GUU from yeast was added to the lysate (Carlson et al 2001). When the effect by Q34 was analyzed in vivo using a Q34 deficient mutant of E. coli, frameshifting by tRNA Leu U?AA decreased 2-fold at the U-UUA-AAC site (i.e., when tRNA Leu U?AA interacted with the AAC codon) and increased 2-fold at the U-UUA-AAU site (i.e., when tRNA Leu U?AA interacted with the AAU codon; Brierley et al 1997).…”

“…Thus, lack of yW37 of tRNA Phe GmAA decoding UUU in the P-site did not influence −1 frameshifting either in vivo or in vitro. Because lack of yW37 of tRNA Phe GmAA increased −1 frameshifting at A-AAU-UUU but not at U-UUU-UUU/C (Carlson et al 1999(Carlson et al , 2001, the yW37-mediated effect on tRNA Phe GmAA decoding UUU in the A-site may be sensitive to which tRNA is occupying the P-site (peptidyl-tRNA Lys versus peptidyl-tRNA Phe ). Taken together, whereas modifications such as ms 2 io 6 A37 and m 1 G37 at position 37 have a profound influence on preventing +1 frameshifting, these modifications and yW37 have no or only a minor effect on −1 frameshift errors.…”

“…Using the rabbit reticulocyte lysate (RRL) in vitro translation system, a threefold increase in −1 frameshifting at the A-AAU-UUU site was attributed to the exchange of yW37 to m 1 G37 in tRNA Phe GmAA , reading UUU in the A-site (Carlson et al 1999(Carlson et al , 2001. However, the level of −1 frameshifting at U-UUU-UUU/C sites, which have codons read by tRNA Phe GmAA both in P-and A-sites, was the same using yW or m 1 G37 containing tRNA Phe GmAA .…”

Discovery of Novel Amidotransferase Activity Involved In Archaeosine Biosynthesis and Structural and Kinetic Investigation of QueF, an Enzyme Involved in Queuosine Biosynthesis

“…(30). Queuosine has been implicated in a number of physiological phenomena including eukaryotic cell proliferation and differentiation (31), tyrosine biosynthesis in mammals (32), translational frameshifts essential to retroviral protein biosynthesis (33,34) and bacterial virulence (35). In eukaryotes queuosine has been further shown to be important in neoplasm staging (36).…”