Nucleotides tRNA modification

A -Threonylcarbamoyladenosine (t A) is a hypermodified nucleotide found in decoding codons of some tRNAs, and the endopeptidase-like kinase chromatin associated receptor (EKC/KEOPS) is a conserved complex involved in many cellular processes in archaea and eukarya. Mutational analysis of EKC/KEOPS revealed a direct role of the eomplex in tRNA modification. A -Methyladenosine has also been shown to be a major factor in fat mass and obesity-associated protein (FTO). As was found with the corresponding A -cytidine derivative, eagjng of adenosine with A -nitrodibenzofuran led to deprotection up to 12 times more efficiently than the more commonly used NPE photo-label, and it was specifically removed by irradiation at 365 nm. ... 

Posttranslational modification of preformed polynucleotides can generate additional bases such as pseudouridine, in which D-ribose is linked to C-5 of uracil by a carbon-to-carbon bond rather than by a P-N-glycosidic bond. The nucleotide pseudouridylic acid T arises by rearrangement of UMP of a preformed tRNA. Similarly, methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP (thymidine monophosphate), which contains ribose rather than de-oxyribose. 

Clearly, the results emerging suggested that at least two nucleotides were modified, the absolute sequence position within the tRNA had yet to be established. Ching etalP showed that a Se U residue was present in the wobble position of the tRNA " from C. sticklandii. This study confirmed a notion that the modification probably affects the translation efficiency of certain transcripts, based on the level of modification by selenium. The authors speculated that the modification to seleno-tRNA (GAG) allowed for more efficient use of this tRNA species as compared to the tRNA (GAA). Even today, no definitive data exist to show that this modification alters the translation efficiency in these bacterial model systems. Nonetheless, these studies had established the chemical forms of Se U and mnm Se U, and established that they were derived from modifications to nucleotides that first required sulfur (S U and mnm S U), the mechanism by which selenium was inserted into the tRNA would not be definitively answered until many years later. 

More detailed single extraction experiments have been run with host modification 71b. Not only dinucleotides could be transferred into the organic phase but also a great variety of nucleotides of different chain length, e.g., d(TA)3, d(As), d(T3G2A2), and even a 76-bases tRNA strand. The only apparent necessity for the guests to be recognized seems to be the presence of at least a few adenines in the strand. 

Figure 28-10 Sequence of an E. coli tyrosine tRNA precursor drawn in a hypothetical secondary structure. Nucleotides found modified in the mature tRNA are indicated with their modifications (S4, 4-thiouridine Gm, 2 -0-methylgua-nosine 1°, N6-isopentenyladenosine jt, pseudouridine T, ribothymidine see also Fig. 5-33).241 A partial sequence of the tRNA gene past the CCA end is also shown. Note the region of local 2-fold rotational symmetry (indicated by the bars and the dot). The anticodon 3 -CUA (shaded) of this suppressor tRNA pairs with termination codon 5 -UAG.

Following synthesis, nucleotides in the tRNA molecule may undergo modification to create unusual nucleotides such as 1-methylguanosine (m G), pseudouridine (4/), dihydrouridine (D), inosine (I) and 4-thiouridine (S4U). 

All classes of RNA transcripts must be processed into mature species. The reactions include several types Nucleolytic cleavage, as in the separation of the mature rRNA species from the primary transcript of RNA polymerase I action Chain extension (non-template-directed), as in the synthesis or regeneration of the common CCA sequence at the 3 end of transfer RNAs or of PolyA at the 3 end of mRNAs and Nucleotide modification, for example, the synthesis of methylated nucleotides in tRNA or rRNA. These reactions are a feature of both prokaryotic and eukaryotic gene expression, and the biological consequences are diverse. For example, modified nucleotides can affect the way in which a tRNA recognizes different codons. 

Fig.4. 13 Blasticidin S. Blasticidin S (purple) base pairs to the P-loop of rRNA (grey). Its guanidinium tail forms hydrogen bonds (dotted lines) to the phosphate and stacks hydrophobically onto the base of Hm A2474, a nucleotide that is protected from chemical modification (green) by blasticidin S. Superimposition of rRNA between crystal structures causes blasticidin S to become superimposed on C74 and C75 (yellow) of the analogue of a CCA end of tRNA. Fig. from [7].

Unique architectural features of tRNAs also can serve as identity elements (2). For example, the long variable loop of IRNA " interacts specifically with SerRS. In addition, the tertiary G15 G48 Levitt base pair in E. coli tRNA , and the triplet interaction in tRNA P, is formed between G45, and the G10 U25 parr confers identity. Occasionally, modified nucleotides can act as determinants, as in the case of coli tRNA , tRNA , tRNA T , and yeast tRNA . All of these tRNAs contain modifications in the anticodon loop. 

Fig. 2. The peptidyltransferase center. The structure of the central loop of Domain V of E. coli 23S rRNA is shown. Nucleotides involved in resistance against different inhibitors are indicated. Closed symbols indicate resistance and open symbols protection against chemical modification by bound antibiotic. Mutations that confer resistance to anisomycin in archaea are indicated [87] (Hcu, Halobacterium cutirubrum Hha, H. halobium). The presence of either a G or U at position 2058 in archaea is also indicated. As a consequence of this change archaea are resistant to erythromycin (Hmo, Halococcus morrhuae, Mva, Methanococcus vannielii Tte, Thermoproteus lenax Dmo, Desulfurococcus wofirfo) [29,30,88,90]. Positions where crosslinking to photoreactive derivatives of Phe-tRNA and puromycin have been observed as well as nucleotides protected by bound tRNA are also indicated. Modified from ref [73].

Two structures incorporating modified nucleotides include the co-crystal structure of pseudouridine synthase TruB with a T stem-loop of tRNA in which the modification site (U55) is modified with 5-fluorouridine, " and the crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to DNA containing an abasic site. " ... 

Unusual nucleotides are produced in mature tRNA by posttranscrip-tional modification of normal nucleotides, and a CCA sequence is added at the 3 end. 

Figure 5-22. Synthesis of tRNA. D, T. y, and are unusual nucleotides produced by posttranscriptional modifications.

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