TRNA sequencing

The amino acid transferred to methionine is senne instead of alanine The senne tRNA sequence that is complementary to the UCU sequence of mRNA is AGA... 

TransferR A. Over 2000 sequences of tRNAs have been deterrnined and several tRNA crystal stmctures have been solved (20). In addition, the overall stmctures, as well as some specific nucleotides within the tRNA sequences, are highly conserved. The secondary stmcture (Fig. 9a) conforms to... 

C13-0105. Transfer-RNA molecules (tRNA) have a set of three bases at their tip that are exposed and can bind their complementary bases. What sequence of bases will be recognized by the following tRNA sequences (a) GAU, (b) AGG, and (c) CCU ... 

The improvements in suppression efficiency that the Dougherty and Schultz groups were able to realize through use of tRNAs derived from different natural tRNA species confirm the importance of the tRNA sequence and structure in the suppression mutagenesis system. The results must be viewed with caution, however, as the efficiency of suppression is subject to other variables and is not fully understood. In different proteins, and at different sites within a protein, the results can be dramatically different. Nevertheless, it appears that use of these improved tRNAs will widen the range of analogs that can be introduced and increase protein yields. 

Figure 29-7 (A) Generalized cloverleaf diagram of all tRNA sequences except for initiator tRNAs numbered as in yeast tRNAae (Fig. 5-30). Invariant bases A, C, G, T, U, and semivariant bases Y (pyrimidine base), R (purine base), H (hypermodified purine base). The dotted regions (a, P, variable loop) contain different numbers of nucleotides in various tRNA sequences. See Rich.179 (B) L form of the yeast phenyl-alanine-specific tRNAphe. The structure is the same as that in Fig. 5-31 but has recently been redetermined at a resolution of 0.20 nm.175 The new data revealed the presence of ten bound Mg2+ ions (green circles) as well as bound spermine (green).

Many tRNA sequences are known today, both for a given codon at a variety of phylogenic levels and for a given species and many different anticodons. Each such category is interesting13,14 for comparative analysis. Phylogenic analysis shows whether tRNA has retained information from prebiotic times, or this information has been lost in the course of evolution. The comparison of different tRNA molecules in a single species may then lead to a reasonably complete reconstruction of the early forms and allow statements about the early evolution of the translation apparatus. 

Fig. 7. Correlation analysis of the repetition of purine in tRNA. A tRNA sequence is divided into triplets, beginning at the S end and in phase with the anticodon. The frequency with which a purine (R) in the first position of the triplet occurs n positions later is counted and plotted against n. The period of three which emerges indicates clearly a triplet structure of the form RNY. The curves show values for the averaged sequences of E. coli and of all tRNAs investigated to date these are compared with that of the master sequence arising from the superposition of all tRNAs. The fact that the correlation is clearer in the master sequence suggests that this may represent a "memory of the earliest phase of evolution.

As a comparison, fluorescent labeling of tRNA with PyC is achieved in one step by the CCA enzyme, and thus is conceptually and technically simpler than labeling of tRNA with proflavin, rhodamine, or Cy3 and Cy5-hydrazides via D residues. However, the fluorescence emission intensity of PyC is not as high as those of the other fluorophores and thus may not be suitable for single-molecule experiments. Nonetheless, enzymatic labeling of tRNA with PyC is easy to implement and should be applicable to all tRNA sequences (both wild type and mutants), which can be generated by in vitro transcription without the requirement for a specific modification or for native tRNA species. 

The rRNA transcription units in E. coli contain some tRNA genes that are transcribed and processed at the time of rRNA transcription (Topic G9). Other tRNA genes occur in clusters of up to seven tRNA sequences separated by spacer regions. Following transcription by the single prokaryotic RNA polymerase, the primary RNA transcript folds up into the characteristic stem-loop structures (Fig. 2) and is then processed in an ordered series of cleavages... 

After synthesis, the pre-tRNA molecule folds up into the characteristic stem-loops structures (Fig. 1) and non-tRNA sequence is cleaved from the 5 and 3 ends by ribonucleases. In prokaryotes, the CCA sequence at the 3 end of the tRNA (which is the site of bonding to the amino acid) is enclosed by the tRNA gene but this is not the case in eukaryotes. Instead, the CCA is added to the 3 end after the trimming reactions by tRNA nucleotidyl transferase. Another difference between prokaryotes and eukaryotes is that eukaryotic pre-tRNA molecules often contain a short intron in the loop of the anticodon arm (Fig. 4). 

This non-canonical fold, established according to chemical and enzymatic structure probing, includes an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleosides are required and are responsible for its cloverleaf structure. In a further study a chimeric tRNA with the sole modification of 1-methyladenosine in position 9 was synthesized it was demonstrated that this chimeric RNA folds correctly [27]. Thus, because of Watson-Crick base-pair disruption, a single methyl group is sufficient to induce the cloverleaf folding of this unusual tRNA sequence. 

Sprinzl M, Gauss DH (1982) Compilation of tRNA sequences. Nucl Acids Res 10 rl-r55... 

Figure 29.3. Alanine-tRNA Sequence. The base sequence of yeast alanyl-tRNA and the deduced cloverleaf secondary structure are shown. Modified nucleosides are abbreviated as follows methylinosine (ml), dihydrouridine (UH2),...

Fig. 2. Example of output formats for the structure predicted for the third tRNA sequence of Fig. 1. This structure is composed of three helices.

Fig. 3. Snapshot of RNAfamily. It shows the common structure for the three tRNA sequences of Fig. 1. Clicking on a stem displays the nucleotidic content of the stem (here the green stems). Clicking on ggau in the left menu displays the nucleotidic content of all sequences.

Fig. 4. Example of combination of caRNAc and Mfold. The first two structures (A,B) are the best two results given by Mfold alone for the third tRNA sequence of Fig. 1. The last structure (C) is obtained with Mfold using constraint information produced by caRNAc (file C in Fig. 2). In this case, Mfold correctly completes the structure and identifies the fourth stem that is missing in caRNAc output. This leads to the typical clover leaf structure (the acceptor stem is on the top).

A fundamental tenet of molecular biology is that the function of biological macromolecules depends on their structure. As a consequence, these structures tend to be more strongly conserved in evolution than their corresponding sequences. In the case of RNA, structure is most accessible on the level of secondary structure, i.e., the pattern of basepairings. A striking example of such structural conservation are the tRNAs, where almost all tRNA sequences, be they from animals, plants, or bacteria, fold into the characteristic cloverleaf secondary structure. Similar structural conservation is found for most of the... 

As a first example, we will produce a consensus structure prediction for the following three tRNA sequences. 

Fig. 1. Consensus structure prediction for three tRNA sequences in different representations. Top row conventional secondary structure drawing as produced by RNAalifold (left) and colorrna.pl (right). Second row dot plot and mountain representation. Bottom alignment with consensus structure in bracket format, and conservation curve as produced by coloraln.pl. In this black and white version, red (no variation) is replaced by light gray, ochre (two types of pairs) by medium gray, and green (three types of pairs) by dark gray. Color versions of all figures can be found in the electronic supplement.

Posttranscriptional processing of tRNA requires several distinct steps, as summarized in Figure 25.8. First, the 50 and 30 ends must be cleaved to release the tRNA sequence from the larger precursor transcript and introns must be removed if they are present. Second, the required CCA charging sequence at the 30 end of tRNA must sometimes be added by a nucleotidyl transferase. Third, all tRNAs contain a large number of modified bases which result from reductions, methylations, and deaminations. These modifications can affect codon recognition by the tRNAs during protein synthesis (Chapter 26). 

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