9.15:
Transfer RNA Synthesis
Transfer RNAs, or tRNAs, are non-coding RNAs that play a major role in protein synthesis. Eukaryotic cells contain more than 50 distinct tRNAs, each carrying a specific amino acid.
The folded tRNA has three hairpin loops: an anticodon loop, a T loop, and a D loop. The 3’ end of the molecule has a conserved CCA sequence that covalently binds an amino acid. Additionally, tRNAs contain many modified bases at several positions.
A tRNA gene is transcribed by RNA Polymerase III as a long precursor tRNA, or pre-tRNA. The pre-tRNA contains a 5’ leader sequence, a 3’ trailer sequence comprising a polyuridine tract, a 14-nucleotide long intron, and unmodified bases.
The precursor tRNA undergoes post-transcriptional processing and modifications before it yields a mature tRNA. The extent of processing varies significantly in order and kind for different tRNAs.
The first step in the tRNA processing involves the removal of the 5’ leader sequence and is catalyzed by an RNA enzyme called Ribonuclease P, or RNase P. This enzyme contains a catalytically active RNA that removes the 5’ leader sequence.
In the second step, the trailer sequence at the 3′ end is trimmed by one or more nucleases, such as the exonuclease RNase D. In the third step of the series, the 3’ terminal trinucleotide CCA, which is missing in some bacterial and all eukaryotic tRNA precursors, is added.
In all eukaryotic pre-tRNAs, an enzyme called tRNA nucleotidyltransferase adds the CCA sequence to the processed 3′ end.
Next, multiple nucleotides in the pre-tRNA are chemically modified at specific positions. Common base modifications include methylation, deamination, reduction, and isomerization.
In the final step of tRNA processing, the intron sequence gets spliced from tRNA transcripts to produce a mature tRNA.
9.15:
Transfer RNA Synthesis
One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with three enzymes dedicated for the methylation of Guanine at different positions.
The nature and position of these modified bases are species-specific. Thus, there are several bases that are exclusive to eukaryotes or prokaryotes. For instance, thiolation of Adenine is only observed in prokaryotes, whereas methylation of cytosine is restricted to eukaryotes. Overall, eukaryotic tRNAs are modified to a greater extent than those from prokaryotes.
Although the nature of modifications may vary, some regions of the tRNA are always heavily modified. Each of the three stem-loop regions or "arms" of the tRNA have modified bases that serve unique purposes. The TΨC arm, named after the presence of the nucleotides, thymine, pseudouridine and cytosine, is recognized by the ribosome during translation. The DHU or D arm that contains the modified pyrimidine dihydrouracil serves as a recognition site for the aminoacyl-tRNA synthetase enzyme, that catalyzes the covalent addition of an amino acid to the tRNA. The anticodon loop often has a queuine base, which is a modified guanine. This base creates a Wobble pair with the codon sequence on the mRNA, i.e. it forms a base pair that does not follow Watson-Crick base pair rules. Usually, a tRNA binds the mRNA more “loosely” in the third position of the codon. This allows several types of non-Watson–Crick base pairing or Wobble bases at the third codon position. It has been observed that the presence of queuine in the first position of the anticodon, which pairs with the third position of the codon, improves the translation accuracy of the tRNA.
Suggested Reading
- Brahmachari, Vani, and T. Ramakrishnan. "Modified bases in transfer RNA." Journal of Biosciences 6, no. 5 (1984): 757-770.
- Crick, F. H. C. "Codon-anticodon pairing: the wobble hypothesis." (1966).