What do you call the three tRNA bases that code for an amino acid

tRNA fragment mediated regulation in lung cancer

Recent studies have suggested dysregulation in tRNA derivatives in lung cancer. These are generated by the specific cleavage at anti-codon loops, TψC loops, D loops, and other positions of pre- and mature tRNAs by nucleases [41]. Pekarskya et al. demonstrated that ts-3676 and ts-4521, which are derived from tRNA-Thr and tRNA-Ser respectively, could serve as microRNAs and piRNAs [42,43]. They were also found to be drastically down-regulated and mutated in lung cancer samples versus matched normal lung tissues. Of note, ts-4521 deletion in cells led to alteration in cell proliferation-related pathway and apoptosis-related pathways. Similarly, overexpression of ts-46 and ts-47 was found to significantly reduce the clonal formation in lung cancer cells, further confirming the involvement of these tsRNAs in lung cancer pathogenesis [44].

V.F Availability of Amino Acids, tRNA Abundance, and Codon Usage

TRDMT1/DNMT2

tRNA aspartic acid methyltransferase 1 (TRDMT1) also known as DNMT2 is a second tRNA (cytosine-5)-methyltransferase that methylates cytosine 38 (C38) in the anticodon loop of several tRNAs using a DNA methyltransferase-like mechanism [114,115]. DNMT2-mediated C38 methylation was shown to protect tRNAs against cleavage by the ribonuclease angiogenin [115]. However, the physiological role of this tRNA methyltransferase remained elusive for long, as DNMT2 mutant mice are viable and fertile. DNMT2 activity appears particularly important for cellular differentiation during hematopoiesis [116]. Here, loss of DNMT2 disrupts differentiation of bone-marrow mesenchymal stem cells and affects specific protein-synthesis fidelity. While a systematic evaluation of DNMT2 expression in human tissues and in cancer patients is still lacking, DNMT2 was found upregulated in hundreds of tumors samples listed in the COSMIC database [117,118]. Moreover, somatic mutations in DNMT2, which affect its activity, were found in tumors [119]. Functional studies addressing the role of this tRNA methyltransferase in cancer, cellular growth, and proliferation are still required to further define the potential contribution of DNMT2 in cancer biology.

tRNA modifications

tRNAs are heavily post-transcriptionally modified, with multiple methylation and pseudouridylation sites clustering within functionally important regions of the particle. These modifications affect tRNAs' localization, interaction with ribosome decoding site and codon:anticodon recognition. In particular, they affect the architecture of the anticodon loop to modulate the wobble interactions. That results in changed tRNA binding affinity to the cognate and near-cognate codons impacting the competition with other tRNAs. Moreover, the hypomodification of tRNAs results in their destabilization by rapid tRNA decay, changing the composition of the tRNA pools within the cell. Now it starts to be evident that modifications positioned on tRNAs are dynamic and changing in different cellular conditions, including cancer. However, importance and meaning of differential tRNA modifications remain unclear and it requires further intensive studies to understand whether they play causative role or are just mute bystanders and the markers of the ongoing oncogenic process.

Several different nucleotide modifications have been described for tRNAs. In particular, quenosine, cytidine 5′methylation (m5C), 5-carbamoylmethylation of uridine (mcm5U) or N6-isopentenylation of adenosine (i6A) are all present in tRNAs, preferentially within anticodon loop at uridine 34. They are also subjected to dynamic changes in cancer. Accordingly, levels of ‘writer’ enzymes (ELP3 and ALKBH8 for mcm5U, NSUN2 for m5C, TRIT1 for i6A and TGTase for quenosine) that position modifications on tRNA are mostly upregulated in variety of cancers, including breast, colorectal, cervical or bladder tumors. Notably, there are no known ‘erasers’ for tRNA modifications and this still an open question if tRNA modifications may be removed. The accepted paradigm at the moment is that pools of tRNAs must be degraded to change the stoichiometry of the modifications, however some reports indicate the reversibility of some of the modifications, such as for the m1A removed by ALKBH1 [59].

Finally, the emerging evidence suggests that not only the full length tRNAs but also derived fragments may undergo the post-transcriptional modifications. It was recently reported that short tRFs containing terminal oligoguanine stretch, termed mTOGs may undergo the dynamic changes in pseudouridylation downstream to stand-alone pseudouridine synthase 7 (PUS7) enzyme [54]. This novel regulatory network balance the translation during embryonic development but also in the subset of pre-leukemic stem cells derived from aggressive 7- myelodysplastic syndrome.

Altogether, tRNAs may be easily (and very simplistically) perceived as simple carriers of amino acids, with little potential for regulation. Instead, it emerges that they are readily tuned, serving as a source of novel regulatory molecules with important role in cancerogenesis.

Tetracycline Resistance

Tetracyclines have been used extensively over the last 50 years to treat a variety of bacterial infections in both humans and animals. The compounds inhibit bacterial cell growth by the inhibition of protein synthesis at the level of the ribosome by disrupting the codon-anticodon interactions between tRNA and mRNA (45). Bacterial resistance to this class of antimicrobial agents is a growing problem and is primarily due to the transfer of resistance genes rather than via spontaneous mutations (46). The exact mechanism of resistance to tetracyclines is not completely understood, but it appears that a cytoplasmic protein associates with the ribosome and allows for protein synthesis to occur even in the presence of bound tetracycline. Like macrolides, the most prevalent mechanism of resistance to tetracyclines is ribosomal protection (14,15). The genes responsible for this are designated Tet(M), Tet(O) and Tet(Q) and typically reside in plasmids and/or transposons (45-47). Tet(M) and Tet(O) are found in a variety of gram-positive and gram-negative organisms such as Staphylococcus spp. while Tet(Q) is found only in Bacteroides (48).

3.2.3 DNA and Gene Expression

DNA (see Figure 3.3) is found in the nucleus and mitochondria of eukaryotic cells. In organisms that reproduce sexually, the DNA in the nucleus contains information from both parents, while that in the mitochondria comes from the organism's mother. In the nucleus, the DNA is wrapped around protein spools, called nucleosomes, and is organized into pairs of chromosomes. Humans have 22 pairs of autosomal chromosomes and two sex chromosomes, XX for females and XY for males (Figure 3.12). If the DNA from all 46 chromosomes in a human somatic cell—that is, any cell that does not become an egg or sperm cell—was stretched out end to end, it would be about 2 nm wide and 2 m long. Each chromosome contains thousands of individual genes that are the units of information about heritable traits. Each gene has a particular location in a specific chromosome and contains the code for producing one of the three forms of RNA (ribosomal RNA, messenger RNA, and transfer RNA). The Human Genome Project was begun in 1990 and had as its goal to first identify the location of at least 3,000 specific human genes and then to determine the sequence of nucleotides (about 3 billion!) in a complete set of haploid human chromosomes (one chromosome from each of the 23 pairs). See Chapter 13 for more information about the Human Genome Project.

DNA replication occurs during cell division (Figure 3.13). During this semiconservative process, enzymes unzip the double helix, deliver complementary bases to the nucleotides, and bind the delivered nucleotides into the developing complementary strands. Following replication, each strand of DNA is duplicated so two double helices now exist, each consisting of one strand of the original DNA and one new strand. In this way, each daughter cell gets the same hereditary information that was contained in the original dividing cell. During replication, some enzymes check for accuracy, while others repair pairing mistakes so the error rate is reduced to approximately one per billion.

Since DNA remains in the nucleus, where it is protected from the action of the cell's enzymes, and proteins are made on ribosomes outside of the nucleus, a method (transcription) exists for transferring information from the DNA to the cytoplasm. During transcription (Figure 3.14), the sequence of nucleotides in a gene that codes for a protein is transferred to messenger RNA (mRNA) through complementary base pairing of the nucleotide sequence in the gene. For example, a DNA sequence of TACGCTCCGATA would become AUGCGAGGCUAU in the mRNA. The process is somewhat more complicated, since the transcript produced directly from the DNA contains sequences of nucleotides, called introns, that are removed before the final mRNA is produced. The mRNA also has a tail, called a poly-A tail, of about 100–200 adenine nucleotides attached to one end. A cap with a nucleotide that has a methyl group and phosphate groups bonded to it is attached at the other end of the mRNA. Transcription differs from replication in that (1) only a certain stretch of DNA acts as the template and not the whole strand, (2) different enzymes are used, and (3) only a single strand is produced.

After being transcribed, the mRNA moves out into the cytoplasm through the nuclear pores and binds to specific sites on the surface of the two subunits that make up a ribosome (Figure 3.15). In addition to the ribosomes, the cytoplasm contains amino acids and another form of RNA: transfer RNA (tRNA). Each tRNA contains a triplet of bases, called an anticodon, and binds at an area away from the triplet to an amino acid that is specific for that particular anticodon. The mRNA that was produced from the gene in the nucleus also contains bases in sets of three. Each triplet in the mRNA is called a codon. The four possibilities for nucleotides (A, U, C, G) in each of the three places give rise to 64 (43) possible codons. These 64 codons make up the genetic code. Each codon codes for a specific amino acid, but some amino acids are specified by more than one codon (Table 3.1). For example, AUG is the only mRNA codon for methionine (the amino acid that always signals the starting place for translation—the process by which the information from a gene is used to produce a protein), while UUA, UUG, CUU, CUC, CUA, and CUG are all codons for leucine. The anticodon on the tRNA that delivers the methionine to the ribosome is UAC, whereas tRNAs with anticodons of AAU, AAC, GAA, GAG, GAU, and GAC deliver leucine.

Table 3.1. The Genetic Code

First BaseSecond BaseThird BaseAUGCALysIleArgThrALysMet - StartArgThrGAsnIleSerThrUAsnIleSerThrCUStopLeuStopSerAStopLeuTrpSerGTyrPheCysSerUTyrPheCysSerCGGluValGlyAlaAGluValGlyAlaGAspValGlyAlaUAspValGlyAlaCCGlnLeuArgProAGlnLeuArgProGHisLeuArgProUHisLeuArgProC

Amino acid 3-letter and 1-letter codes: Ala (A) = Alanine; Arg (R) = Arginine; Asn (N) = Asparagine; Asp (D) = Aspartic acid; Cys (C) = Cysteine; Glu (E) = Glutamic acid; Gln (Q) = Glutamine; Gly (G) = Glycine; His (H) = Histidine; Ile (I) = Isoleucine; Leu (L) = Leucine; Lys (K) = Lysine; Met (M) = Methionine; Phe (F) = Phenylalanine; Pro (P) = Proline; Ser (S) = Serine; Thr (T) = Threonine; Trp (W) = Tryptophan; Tyr (Y) = Tyrosine; Val (V) = Valine.

During translation, the mRNA binds to a ribosome and tRNA delivers amino acids to the growing polypeptide chain in accordance with the codons specified by the mRNA. Peptide bonds are formed between each newly delivered amino acid and the previously delivered one. When the amino acid is bound to the growing chain, it is released from the tRNA, and the tRNA moves off into the cytoplasm, where it joins with another amino acid that is specified by its anticodon. This process continues until a stop codon (UAA, UAG, or UGA) is reached on the mRNA. The protein is then released into the cytoplasm or into the rough ER for further modifications.

Example Problem 3.5

Consider a protein that contains the amino acids asparagine, phenylalanine, histidine, and serine in sequence. Which nucleotide sequences on DNA (assuming that there were no introns) would result in this series of amino acids? What would be the anticodons for the tRNAs that delivered these amino acids to the ribosomes during translation?

Transfer RNA Covalently Binds Amino Acids and Recognizes Specific Regions of mRNA

How does mRNA specify the sequence of amino acids in a protein? Which amino acid is to be incorporated into the protein is specified by a sequence of three nucleotides called a codon. The mRNA triplets do not directly recognize and specify the amino acids; they do so through the use of another kind of RNA called transfer RNA or tRNA. These remarkable molecules are adapters that can link with an amino acid and recognize the triplets of nucleotides on the mRNA, the codons. They do this by containing a sequence complementary to the codon: the anticodon. The function that maps triplets of nucleotides on the mRNA to specific amino acids is called the genetic code. Figure 2.2.5 shows the genetic code in look-up table format.

The tRNA consists of a single strand of RNA from 70 to 90 nucleotides long that is held together by hydrogen bonding within nucleotides on the same chain. One end of the tRNA allows for covalent attachment of an amino acid. Another section of the tRNA contains a sequence of three nucleotides that forms the anticodon. Precursors to the tRNA are transcribed from DNA by RNA polymerase III.

Another key in the formation of proteins is the attachment of amino acids to the specific tRNA. Specific enzymes called aminoacyl-tRNA synthetases couple the amino acid to the appropriate tRNA. There is a different synthetase for each amino acid. One attaches glycine to tRNAGly, another attaches alanine to tRNAAla, and so on. These synthetases must recognize both the amino acid and the tRNA that contain the right anticodon. The overall processing of RNA and protein synthesis is shown in Figure 2.2.6. Translation is shown in Figure 2.2.7.

4.1.3 Translation

The genetic information transcribed into the mRNA is expressed by means of a triplet code which is translated into a polypeptide chain by the transfer RNA (tRNA). In this code, three bases on the mRNA form a coding group (known as a codon) which codes for a specific amino acid. The tRNA has a loop comprising three nucleotides with bases complementary to those of the codon (known as an anticodon) and, in addition, an acceptor arm which binds with the amino acid specific to that anticodon. Each tRNA functions to transport its bonded amino acid to the ribosomal RNA (rRNA),10 the structural component of the ribosomes, for incorporation into the polypeptide chain.

The theory of the triplet code was deduced from the number of amino acids that could be coded for by four bases. Fewer than three bases would not be enough to code for 20 amino acids,11 so the minimum number of bases had to be three. This, however, would theoretically provide 64 combinations, which means that there is some redundancy. The redundancy has been accounted for in that various codons are known to code for the same amino acid12 and that some codons are used for punctuation to initiate or terminate an amino acid sequence.

Even before the mRNA has been completely transcribed from the DNA in the nucleus, the genetic information in the mRNA is translated into a polypeptide chain on the ribosomes (Figure 4.1). A start codon on the mRNA signals the initiation of translation with the first amino acid (usually AUG13 which codes for the amino acid methionine). The mRNA is aligned with the P (peptidyl) site on the ribosome and the tRNA is bound to this P site and to the mRNA by hydrogen bonding between the codon of the mRNA and its anticodon. The unoccupied adjacent A (aminoacyl) site is aligned with the next codon of the mRNA and the anticodon of the next tRNA binds to the A site and to the codon of the mRNA in the site. The first amino acid is then transferred from its tRNA to form a peptide bond with the second amino acid. The mRNA advances one codon and the tRNA that was previously located at the P site is moved to the E (exit) site, from where it is released. The tRNA that was formally bonded to the A site now moves into the P site. The A site becomes free to attract another tRNA and the process is repeated. Elongation of the polypeptide chain continues 14 until the stop codon of the mRNA (usually UAA, UGA or UAG)15 enters the A site, signifying the termination of translation. The polypeptide chain is released and the ribosome dissociates from the mRNA.

In this way, the genetic information contained in the DNA and transcribed into the mRNA translates into a specific polypeptide and hence ultimately defines the functioning and regulation of the cell.

The sequence of DNA nucleotides that code for a specific polypeptide is called a gene. Adjacent genes are generally transcribed into one mRNA that may code for more than one polypeptide.