Avatar pre-tRNAs help elucidate the properties of tRNA-splicing endonucleases that produce tRNA from permuted genes

Abstract

Unusual tRNA genes, found in some algae, have their mature terminal 3' portion in front of their 5' portion in the genome. The transcripts from such genes must be cleaved by a pre-tRNA endonuclease to form a functional tRNA. We present a mechanism for the generation of "corrected" tRNAs from such a "permuted" pre-tRNA configuration. We used two avatar (av) or model pre-tRNAs and two splicing endonucleases with distinct mechanisms of recognition of the pre-tRNA. The splicing results are compatible with an evolutionary route in which permuted genes result from a duplication event followed by DNA rearrangement. The model pre-tRNAs permit description of the features that a transcript, derived from a rearranged duplicated gene, must have to give rise to functional tRNA. The two tRNA endonucleases are a eukaryal enzyme that normally acts in a mature domain-dependent mode and an archaeal enzyme that acts in a mature domain-independent mode. Both av pre-tRNAs are able to fold into two conformations: 1 and 2. We find that only conformation 2 can yield a corrected functional tRNA. This result is consistent with contemporary algae representing snapshots of different evolutionary stages, with duplicated genes preceding recombinatorial events generating a permutated gene. In a scenario elucidated by the use of the av pre-tRNAs, algal permuted tRNA genes could have further lost one of two mature domains, eliminating steric problems for the algal tRNA endonuclease, which remains a typical eukaryal enzyme capable of correcting the permuted transcript to a functional tRNA.

Fig. 1.

Predicted structures of possible conformation of the duplicate pre-tRNABHB (FA*FB). Each Pre-tRNA^BHB consists of two regions derived from yeast pre-tRNA^Phe (nucleotides 1–31 and 38–76), joined by a 25-nt insert that corresponds to the BHB motif of archaeal tRNA^Trp; the sequence is written in capital letters. The 5′ halves and the 3′ halves are colored in gray and the intron in black. The leader sequence, the trailer, and the spacer sequence are written in lowercase letters. A rectangular box delimits the spacer sequence. Triangles indicate the splicing sites for METJA and SCHPO tRNA endonuclease; black arrows indicate the size of the two synthetized transcripts (FA and FA*FB). The products of the reactions and the size in nucleotides are indicated.

Fig. 2.

Predicted structures of possible conformation of the duplicate pre-tRNABHB (FMA*FMB). tRNA FMA is a mutant tRNA FA lacking the 5′ bulge and FMB is a mutant tRNA FB lacking the 3′ bulge. Gray triangles indicate the splicing sites for both METJA and SCHPO tRNA endonuclease; the white triangle indicates the splicing site of the SCHPO tRNA endonuclease. The black arrows indicate the size of the two synthetized transcripts (FMA and FMA*FMB). The products of the reactions and the size in nucleotides are indicated.

Fig. 3.

Processing by METJA tRNA endonuclease. Lanes: 1, pre-tRNA^BHB; 2, FA*FB; 3, FMA*FMB; 4, FMA; 5, FA; 6, pre-tRNA^BHB; 7, tRNA^Phe. The reaction products are indicated.

Fig. 4.

Processing by SCHPO tRNA endonuclease. Lanes: 1, FMA; 2, FA; 3, FMA*FMB; 4, FA*FB; 5, pre-tRNA^BHB; 6, pre-tRNA^BHB; 7, tRNA^Phe. The reaction products are indicated.