Trbp111 selectively binds a noncovalently assembled tRNA-like structure

Abstract

Transfer RNAs are key components of the genetic code by virtue of aminoacylation reactions whereby each amino acid is linked to the tRNA that bears the anticodon for the attached amino acid. The L-shaped tRNA structure contains two domains connected at right angles through a corner formed from tertiary interactions involving loops of each domain. Some evidence suggests that the domains arose separately and eventually were fused into a single covalent structure. In this scenario, the present-day tRNA possibly developed through a noncovalently assembled heterodimeric intermediate. Trbp111 is an ancient structure-specific tRNA binding protein that interacts specifically with the outside corner of the L-shaped molecule. Plausibly, this protein could act as a chaperone to cover and protect the fragile corner and thereby have a historical role in the development of tRNA. Here we show that Trbp111 interacts with a noncovalently assembled tRNA-like structure, under conditions where it does not interact with individual tRNA domains. Trbp111 binding specifically requires formation of the tRNA-like corner. In a mixture of RNA domains, it selects those that can make the L-like structure. Thus, cofactors such as Trbp111 have the capacity to help assemble and stabilize RNA dimers that are tRNA-like.

Fig 1.

Establishment of L-shaped tRNA through noncovalent association of the two domains. (a Upper) The tRNA cloverleaf and its rearrangement into an L-shaped structure with tertiary interaction common to all tRNAs shown with dashed lines. (a Lower) Noncovalent assembly of RNA domains to make a tRNA-like structure. Trbp111 dimer is shown as gray spheres. Base pairing between loops is shown with dashed lines. (b) Sequence of the RNA minihelix and anticodon SBL used in the experiments. The 7-nt loop and the first two base pairs of the closing stem from RNA I and RNA II (12) are highlighted in pale gray.

Fig 2.

Formation of the RNA dimer complex through complementary loop–loop interactions. A gel retardation assay was carried out at pH 7, 4°C, with 5′-³²P-labeled minihelix 35-mer (≈1 nM) in which unlabeled anticodon SBL was added in increasing concentrations from 0 to 15.5 μM. The shifted band was quantified and plotted on a logarithmic scale to obtain the binding profile shown on the left. The corresponding Hill plot is shown on the right.

Fig 3.

Mutation in the loop sequence disrupts complex formation. (a) Sequence of the loop used for minihelices (mut-3) and (mut-5). The potential base pairing interactions are shown with dotted lines. (b) Gel retardation assay using mutant forms of minihelix. Each 5′-³²P-labeled minihelix (≈900 nM) was incubated with unlabeled anticodon SBL for 30 min at room temperature before loading on the gel. (c) Competition assay using unlabeled minihelices. Each unlabeled minihelix was added in increasing concentrations from 10 nM to 10 μM into the mixture of 5′-³²P minihelix (≈1 nM) and unlabeled anticodon SBL (1.6 μM).

Fig 4.

Gel retardation assay with Trbp111. Anticodon SBL was ³²P-labeled and added with indicated RNA species. The identity of each shifted band is denoted on the left.

Fig 5.

Binding profile of Trbp111 to the RNA dimer complex. Trbp111 was added in increasing concentrations to the preformed RNA dimer with 5′-³²P-labeled minihelix (≈900 nM) and unlabeled anticodon SBL (16 μM). The band corresponding to the ternary complex was quantified and plotted on a logarithmic scale to obtain the binding profile shown below.

Fig 6.

(a) Mutation in the loop sequence disrupts ternary complex formation. 5′-³²P-labeled anticodon SBL (≈850 nM) was incubated with Trbp111 (37 μM) and unlabeled minihelix and minihelix (mut-3) in increasing concentrations from 100 nM to 100 μM. (b) Competition assay using unlabeled minihelices. 5′-³²P minihelix was incubated with unlabeled anticodon SBL (20 μM) and Trbp111 (1.9 μM) to which unlabeled minihelix or minihelix (mut-3) was added in increasing concentrations from 100 nM to 100 μM.

Fig 7.

Trbp111 protection from S1 nuclease cleavage. 5′-³²P-labeled minihelix (≈1 nM) was incubated with indicated RNA species. Anticodon SBL (1.6 μM), Trbp111 (43 μM), and S1 nuclease (≈1 unit) were used.

Fig 8.

S82A mutant Trbp111 disrupts binding to the RNA dimer. 5′-³²P-labeled anticodon SBL (≈300 nM) was added with unlabeled minihelix (1.6 μM) and Trbp111 or S82A Trbp111 at indicated concentrations.