Structural basis of reverse nucleotide polymerization

Abstract

Nucleotide polymerization proceeds in the forward (5′-3′) direction. This tenet of the central dogma of molecular biology is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand synthesis where reverse polymerization (3′-5′) would present a "simpler" solution. Interestingly, reverse (3′-5′) nucleotide addition is catalyzed by the tRNA maturation enzyme tRNA His guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNA His guanylyltransferase-tRNA His complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme's active site from the opposite direction (180°flip) compared with similar nucleotide substrates of canonical 5′-3′ polymerases, and the finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that reverse polymerization appeared early in evolution and resembles a mirror image of the forward process.Thg1-tRNA complex | crystal structure | tRNA editing

5′‐End sequencing in Saccharomyces cerevisiae offers new insights into 5′‐ends of tRNAHis and snoRNAs

“…This added G À1 residue serves as an essential identity element for efficient aminoacylation of tRNA His by histidyl-tRNA synthetase (HisRS) [4][5][6][7].Thg1 homologues known as Thg1-like proteins (TLPs) are found in all three domains of life and are biochemically distinct from Thg1 [1,[8][9][10]. Although Thg1 and TLPs share the same overall structure and mechanism of 3 0 -5 0 nucleotide addition, TLPs exhibit a strong preference for adding WC base-paired nucleotides to RNA substrates [8][9][10][11][12][13]. Moreover, in many of the archaeal and bacterial species that encode TLPs, G À1 can be obtained through an alternative post-transcriptional mechanism.…”

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase

“…Thg1 is evolutionarily related to canonical polymerases. [1][2][3][4] All Thg1 homologs encode the conserved catalytic palm domain, [4][5][6][7] which they use to catalyze a single nucleotide addition to the 5' end of tRNA His . Some Thg1 enzymes are capable of catalyzing extended and template dependent nucleotide addition and are, thus, bona fide reverse nucleotide polymerases.…”

“…Some Thg1 enzymes are capable of catalyzing extended and template dependent nucleotide addition and are, thus, bona fide reverse nucleotide polymerases. 1,2,6,8 In eukaryotes, Thg1 is an essential enzyme in tRNA maturation. 9 Thg1 adds a single guanine residue (G ¡1 ) to the 5'-end of premature tRNA His 1,10 .…”

“…The mechanism of reverse polymerization was revealed in complex structures of Thg1 bound to tRNA. 6,7 The co-crystal structures of Thg1 in complex with its substrate tRNA and ATP or GTP showed surprising structural similarity to forward DNA and RNA polymerases despite no obvious conservation of the amino acid sequence between Thg1 and other canonical polymerases. [4][5][6][7] Like its forward polymerase relatives, each Thg1 subunit folds into a hand-shape containing a catalytic palm domain, and substrate binding finger/thumb domains (Fig.…”

“…In both Thg1-tRNA and T7-DNA complexes, the direction of substrate approach to the catalytic core is related to the direction of polymerization. 6,7 tRNA substrates enter Thg1's active site from the opposite side that DNA and RNA enter other canonical 5'-3' polymerases. Furthermore, the finger and thumb domains of Thg1 are on the opposite sides of the catalytic core compared to canonical polymerases.…”