Mali Illangasekare

A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis.

A 29-nt RNA catalyst successively forms the aminoacyl ester phe-RNA, and then peptidyl-RNA (phe-phe-RNA), given phenylalanine adenylate (phe-AMP) as substrate. Catalysis of two related reactions at similar rates supports the argument that RNA catalysts would evolve as groups with similar mechanisms. In particular, successive aminoacyl- and peptidyl-RNA synthesis by one RNA suggests that uncoded but RNA-catalyzed peptide synthesis would evolve before the synthesis of coded peptides.

Affinity selection-amplification from randomized ribooligonucleotide pools.

Publisher Summary Selection-amplification introduced a new capability to the study of RNA and DNA. One could ask if any nucleic acid (less than a certain size) existed that could perform a particular biochemical function and recover that molecule for further study, along with its closely related functional relatives. Such exhaustive investigation of nucleic acid capabilities was unprecedented. This chapter discusses some of the methods and considerations required to carry out the purification, potentially ≈10 14 -fold, of a new RNA from a randomized pool of initial sequences. RNAs are fractionated (selected by affinity chromatography in this chapter) and the selected fraction is converted to complementary DNA (cDNA) that is amplified by polymerase chain reaction (PCR). Finally the DNA from the PCR is transcribed and the cycle is repeated. After the desired activity is observed in the pool or when selection has apparently succeeded, RNAs are cloned and sequenced. Individual clones are then characterized by appropriate structural and functional biochemical assays.

Phenylalanine-binding RNAs and genetic code evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The KD for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10−11. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.

Chiral histidine selection by D-ribose RNA

The invariant choice of L-amino acids and D-ribose RNA for biological translation requires explanation. Here we study this chiral choice using mixed, equimolar D-ribose RNAs having 15, 18, 21, 27, 35, and 45 contiguous randomized nucleotides. These are used for simultaneous affinity selection of the smallest bound and eluted RNAs using equal amounts of L- and D-His immobilized on an achiral glass support, with racemic histidine elution. The experiment as a whole therefore determines whether RNA containing D-ribose binds L-histidine or D-histidine more easily (that is, by using a site that is more abundant/requires fewer nucleotides). The most prevalent/smallest RNA sites are reproducibly and repeatedly selected and there is a four- to sixfold greater abundance of L-histidine sites. RNAs chiral D-ribose therefore yields a more frequent fit to L-histidine. Accordingly, a D-ribose RNA site for L-His is smaller by the equivalent of just over one conserved nucleotide. The most prevalent L-His site also performs better than the most frequent D-His site-but rarer D-ribose RNAs can bind D-His with excellent affinity and discrimination. The prevalent L-His site is one we have selected before under very different conditions. Thus, selection is again reproducible, as is the recurrence of cognate coding triplets in these most probable L-His sites. If our selected RNA population were equilibrated with racemic His, we calculate that L-His would participate in seven of eight His:RNA complexes, or more. Thus, if D-ribose RNA were first chosen biologically, translational L-His usage could have followed.

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

7 Aminoacyl-tRNA Synthetases and Self-acylating Ribozymes

The aminoacyl-tRNA synthetases (aaRS) are at the heart of modern translation, catalyzing the accurate biosynthesis of aminoacyl-tRNAs (aa-tRNAs), the immediate precursors for encoded peptides. However, the first catalysts that made aa-RNAs for coded protein synthesis probably appeared long before any protein aaRS, to serve a preexisting translation system (see below). It presently seems likely that this ancestral translation system relied on a molecule like RNA, and the proto-aaRS catalysts may themselves have been RNAs. As an exercise in the limits of RNA catalysis, and as data relevant to the existence of such an RNA World, we would like to know how closely RNA catalysis can approximate the essential capabilities of the modern aaRS. Below is, first, a brief introduction to these complex modern proteins, then similarly a description of the presently known self-acylating RNAs, and finally an assessment of the question of possible resemblance. AMINOACYL-tRNA SYNTHETASE EVOLUTION Current aaRS are uniformly large proteins (Schimmel and Soll 1979) and therefore could not have existed before translation itself. In fact, because aaRS share essential structures, like the nucleotide-binding Rossman fold, among themselves and with other proteins (Eriani et al. 1990), they presumably were derived from yet more ancient common ancestors. Thus, most aaRS cannot even have been among the first proteins. Aminoacyl-tRNA synthetases from all species can be split into two equally sized protein families, termed type I and type II proteins, characterized by two different sets of conserved amino acid motifs in the active sites for amino acid activation and aa-tRNA...

Small aminoacyl transfer centers at GU within a larger RNA.

Separate aminoacyl transfer centers related to the small …GUNNN..: NNNU ribozyme seem possible at the frequent GU sequences dispersed throughout an RNA tertiary structure. In fact, such activity is easily detected and varies more than 2 orders in rate, probabably being faster at sites with less structural constraint. Analysis of a particular constrained active site in an rRNA transcript suggests that its difficulty lies not in substrate strand association, but in binding and/or group transfer from the aminoacyl precursor. Efficient aminoacyl transfer requires accurate complementarity between large or small ribozymes and oligoribonucleotide substrates, even when only three or four base pairs link the two. Thus, multi-site active ribozymal superstructures might have coordinated an RNA metabolism, including aiding an early translation apparatus.