Initiator tRNA
Created by Christa Doerwaldt
E. coli initiator tRNA is an asymmetric, L-shaped, single-strand unit comprised of nucleic acids with some internal helical structure which features a major and a minor groove, similar to DNA’s double helical structure. It has a total weight of 24833.10 Daltons and a length of 77 residues. It is found in Escherichia coli and is used for the initiation of protein synthesis. It has a relatively high 64.94% GC content, which confers some stability to its structure. Its sequence is: CGCGGGGTGGAGCAGCCTGGTAGCTCGTCGGGCTCATAACCCGAAGATCGTCGGTTCAAATCCGGCCCCCGCAACCA
(24 G’s and 26 C’s in 77 residues total).1
It is similar to other tRNA’s in its 5’ terminal phosphate group and cytosine-cytosine-adenosine “tail” at its 3’ end (which aids in it being recognized as tRNA), which is OH-linked (at its 3’ end). It also shares a “cloverleaf” stem-and-loop secondary structure with four arms (Figure 1, images tab): the “D arm,” “T arm,” anticodon arm and acceptor stem (formed by the pairing of the 5’ and 3’ ends of the molecule). The “D arm” stem is comprised of four base pairs ending in a loop, named as such because that loop contains dihydrouridine, designated U17a (the 77-residue molecule is therefore only labeled 1-76). The “T arm” stem is five base pairs long and its loop contains the characteristic TΨC sequence (thymidine-pseudouridine-cytosine). The anticodon arm contains the methylated Cm32•A38 base pair extending the anticodon helix and an “anticodon” sequence corresponding to the AUG initiation codon, which helps distinguish it from E. coli elongator tRNA.
Together these arms form an “L”-shaped tertiary structure in which the TΨC and acceptor stems form one leg and the anticodon and D stems form the other. This is enabled by coaxial “stacking” of adjacent base pairs and allows it to fit well with the the A and P sites of the ribosome.2 The A and P sites are two of three (A, P and E) RNA binding sites on the ribosome, where “A” indicates the site binds an amino acid-bound tRNA (aminoacyl tRNA) and “P” designates that it binds a peptidyl tRNA, in this case the E. coli initiator tRNA, which is bound to the peptide being synthesized.
As a prokaryotic tRNA, E. coli initiator tRNA differs from eukaryotic tRNA in that it is utilized as formylmethionyl-tRNA (fMet-tRNA) in prokaryotes like E. coli, versus eukaryotes’ use of it without formylation, as methionyl-tRNA (MettRNA).3 Formylation of E. coli initiator tRNA is followed by commitment to the P-site of its respective ribosome via the recruitment of initiator factor 2 (IF2). This is catalyzed by methionyl-tRNAfMet formyltransferase (formylase) and also prevents it from complexing with molecules other than IF2.4 It is also of interest that the inactivation of the formylase gene on the Escherichia coli chromosome severely impairs cell growth.5 Neither the D not the T loops are involved in this interaction.6 In addition, E. Coli initiator tRNA shares with other prokaryotic tRNA's a purine 11: pyrimidine 24 base pair instead of a pyrimidine 11:purine 24 base pair in the D stem as well as a lack of a Watson-Crick base pair at the end of its acceptor stem.
E. coli initiator tRNA has a homolog in yeast (Saccharomyces cerevisiae) initiator tRNA (PDB ID 1YFG), whose sequence is AGCGCCGUGGCGCAGUGGAAGCGCGCAGGGCUCAUAACCCUGAUGUCCUCGGAUCGAAACCGAGCGGCGCUACCA.7 They have 74% sequence homology with a two-residue difference due to C16’s and C17’s (two cytosines’) absence in yeast initiator tRNA, which is 75 nucleic acids long to E. coli initiator tRNA’s 77 nucleic acid residues.8 Where E. coli initiator tRNA has 64.9 % GC content, yeast initiator tRNA has 65.3% GC content which means the triple hydrogen bonds of the G•C pairs adds a comparable amount of stability in both structures. In addition, each has the three GC base pairs in its anticodon stem that characteristically differentiates initiator tRNA’s from other tRNA’s in both prokaryotic and eukaryotic organisms.6 Both have an asymmetric L-shaped tertiary structure and a “cloverleaf” secondary structure. Functionally, the initiator tRNA’s are similar in that both are involved in the initiation of protein synthesis. Also of interest is the high (around 80%) sequence homology between E. coli initiator tRNA and spinach chloroplast initiator tRNA.9 Though the sequence of spinach chloroplast initiator tRNA has not been established for certain (and it does not have a PDB entry) it is worth noting that the observed similarities between E. coli initiator tRNA and spinach chloroplast initiator tRNA point to common evolutionary roots between the plant’s chloroplasts and E. coli bacteria.
Similar to other tRNA’s such as yeast tRNAPhe (or phenylalanine-tRNA), E. Coli initiator tRNA has the reverse Hoogsteen base pairs (meaning one base is rotated 180o with respect to the other) s4U8•A14 and T54•A58 where the “s4U8” indicates a 4-thiouridine modification at position 8. It is also similar in its trans Watson-Crick G15•C48 (Levitt) base pair, which stacks with the U8•A14 reverse Hoogsteen pair to provide stability.10 The one bond imino-4-carbonyl G18•Ψ55 (where Ψ indicates a pseudouridine) and Watson–Crick pair G19–C56 at the corner of the L also stack. The final stacking set is more complex and involves the Watson– Crick-like G26•A44 pair and the U33 ‘U-turn’ in the anticodon loop as well as the base triples A46•(G22–C13) and G45•(G10–C25) and A37• (G29-C41). This stabilizes the sharp turns the molecule makes at its crook as well as the variable loop’s position at the D-stem. One difference here is that the A9•(A23-U12) base triple found in yeast tRNAPhe was not formed in E. Coli initiator tRNA at its equivalent G9•(C23-G12) location but rather absent and replaced by a 5.2-Å distance between G9 and C23.11
The crystal packing of E. Coli initiator tRNA is of interest because its acceptor arm, in which C1 is mismatched with and therefore does not bind to A72 (and also is not face-to-face with A72), will interact with another tRNA molecule to form a triple-helix structure where they meet (Figures 2a and 2b, images tab). In addition, its anticodon loop will interact with another “symmetry-related” tRNA molecule’s anticodon loop, both at their C34A35U36A38 base pairs, forming two typical Watson-Crick AU base pairs and two less common CA pairs. This interaction has also been observed in yeast initiator tRNA (PDB ID 1YFG) and is conjectured to play a role in stabilization of the anticodon loop formation.
In vivo, E. Coli initiator tRNA is bound by Prokaryotic Initiation Factor 2 (IF2),12 which promotes binding of E. Coli initiator tRNA to the E. coli ribosome. It also interacts with peptidyl tRNA hydrolases, which cleaves the connection between the tRNA and the protein being synthesized. Finally, it forms a complex with E. Coli methionyl-tRNAfMet transformylase, which fits into the “L” shape’s D stem side (away from the anticodon stem and loop) and inserts an enzyme loop into the major groove of the acceptor stem’s helix, splitting the C1-A72 (mismatched) pair and causing the 3’ arm to bend inside the active center. The tRNA, once formylated, is prevented from binding to elongation factor (EF) Tu, which binds the acceptor arm of elongator tRNA’s on the T-stem side, and thereby committed to binding to IF2 followed by commitment to the P site of the ribosome.5 Experimentally, Barraud examined E. Coli initiator tRNA at both pH 4.6 (PDB ID 3CW5) and pH 8.0 (PDB ID 3CW6). The main difference is that the “wobble base pair” of C32•A38 (see Figure 3, images tab) is thought to form at pH 4.6 and not at pH 8.0 due to a small change in conformation; however, overall the conformations are nearly identical.