3 -Terminus, of tRNA

Amino-3 -deoxyadenosine. 3 -Amino-3 -deoxyadenosine (17) is elaborated by Cordyceps militarise Aspergillus nidulanSe and Helminthosporium (3,4). The biosynthesis proceeds direcdy from adenosine. Compound (17) inhibits RNA polymerase, but not DNA polymerase, and replaces the adenosyl residue at the 3 -terminus of tRNA. Phenylalanyl-(3 -amino-3 -deoxyadenosyl)-tRNA has acceptor but not donor activity (31,32). Compound (17) also inhibits retroviral RNA-dependent DNA polymerase (33). 

After the proper 5 terminus has been created, ribonuclease D removes the remaining two nucleotides from the 3 end. Should excessive "nibbling" occur through faulty control of RNase D activity, there is an enzyme that will restore the CCA end to any tRNA in a nontranscriptive fashion. This enzyme specifically recognizes the 3 terminus of tRNAs that lack the CCA end and catalyzes sequential reactions with a CTP, another CTP, and an ATP. 

F. Flansske and F. Cramer, Modification of the 3 terminus of tRNA by periodate oxidation and subsequent reaction with hydrazines, Methods Enzymol., 59 (C) (1979) 172-181. 

Transfer RNA (tRNA) serves as a carrier of amino acid residues for protein synthesis. Transfer RNA molecules also fold into a characteristic secondary structure (marginal figure). The amino acid is attached as an aminoacyl ester to the 3 -terminus of the tRNA. Aminoacyl-tRNAs are the substrates for protein biosynthesis. The tRNAs are the smallest RNAs (size range—23 to 30 kD) and contain 73 to 94 residues, a substantial number of which are methylated or otherwise unusually modified. Transfer RNA derives its name from its role as the carrier of amino acids during the process of protein synthesis (see Chapters 32 and 33). Each of the 20 amino acids of proteins has at least one unique tRNA species dedicated to chauffeuring its delivery to ribosomes for insertion into growing polypeptide chains, and some amino acids are served by several tRNAs. For example, five different tRNAs act in the transfer of leucine into... 

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

Figure 12.5 The structures for four tRNA molecules of yeast, (a) Alanyl-tRNA (b) phenylalanyl-tRNA (c) seryl-tRNA (d) tyrosyl-tRNA. The single letter designations identify the sequence of bases along the single chain. Note that several of these are unusual bases, most of which are methylated (Me). Note also the ACC sequence at the 3 terminus of each tRNA. This is the site to which amino acids are attached in the process of protein synthesis, as indicated. These tRNA molecules have a substantial amount of secondary structure created by formation of Watson-Crick base pairs. Finally, note that the anticoding triplet in the bottom loop is shown.

At the ribosome, which travels along the mRNA, the tRNA molecule is bound such that its anticodon can interact with a nucleotide triplet on mRNA (the codon). If the anticodon is complementary to a codon triplet on the mRNA, the amino acid attached at the 3 -terminus of the tRNA is transferred to the amino terminus of the growing polypeptide chain if it is not complementanty, the tRNA is rejected and another one is checked for complementary. The whole process is repeated until the synthesis of the protein is completed. It is initiated, as well as terminated, by specific codons regulating this translation. 

A hybrid dinucleotide pdCpA has been chemically aminoacylated with L-phenylalanine to give pdCpA-Phe. The aminoacylated dinucleotide can be used in an enzymatic ligation (T4 RNA llgase) and attached to the 3 -terminus of a truncated (3 -terminal CA residues missing) amber suppressor tRNA y. The resultant chemically aminoacylated tRNA can be used to incorporate L-phenylalanine in response to an amber nonsense coding. This procedure can... 

Robert Holley first determined the base sequence of a tRNA molecule in 1965, as the culmination ul 7 years of effort, Indeed, his study of yeast alanyl-tRNA provided the first complete sequence of any nucleic acid. This adapter molecule is a single chain of 76 ribonucleotides (Figure 30.2). The 5 terminus is phosphorylated (pCi), whereas the 3 terminus has a free hydroxyl group. T he amino acid-attachment site is the 3 -hydroxyl group of the adenosine residue at the 3 terminus of the molecule. The sequence 5 - IGC-3 in the middle of the molecule is the anticodon, where I is the purine base inosine. It is complementary to 5 -GCC-3, one of the codons for alanine. 

FIGURE 12.6 The aminoacyl-tRNA synthetase reaction, (a) The overall reaction. Everpresent pyrophosphatases in cells quickly hydrolyze the PPj produced in the aminoacyl-tRNA synthetase reaction, rendering aminoacyl-tRNA synthesis thermodynamically favorable and essentially irreversible, (b) The overall reaction commonly proceeds in two steps (i) formation of an aminoacyl-adenylate and (ii) transfer of the activated amino acid moiety of the mixed anhydride to either the 2 -OH (class I aminoacyl-tRNA synthetases) or 3 -OH (class II aminoacyl-tRNA synthetases) of the ribose on the terminal adenylic acid at the 3 -OH terminus common to all tRNAs. Those aminoacyl-tRNAs formed as 2 -OH esters undergo a transesterification that moves the aminoacyl group to the 3 -OH of tRNA Only the 3 -esters are substrates for protein synthesis. 

Photochemical cross-linking studies of ribosomal RNA and a tRNA derivative have been achieved using the 5 -[Y- P]-labelled 3 , 5 -bisphosphate of 2, 6-diazido-9-(P-D-ribofuranosyl)purine. The compound was prepared by reaction of the corresponding nucleoside with pyrophosphoryl chloride followed by exchange of the 5 -phosphate with the y-phosphate of [ y- 2p]j jp catalysed by T4 polynucleotide kinase. It was then enzymatically ligated to the 3 -terminus of a yeast tRNA he molecule. 

The functional roles of rRNAs include the participation in mRNA selection, tRNA binding, ribosomal subunit association, frame-shift snppression, translational proofreading, binding of various factors and peptidyl transferase activity. Messenger RNA is selected by interaction with the 3 -terminus of 16S rRNA in the platform region of the small subunit. tRNA interacts with 16S rRNA in the cleft of the small subunit and around the universally conserved central loop of 23 S rRNA (via CCA terminal of tRNA) of the large subunit. This conserved region of rRNA may be involved in the peptidyl transferase activity. Subunit interaction appears to involve elements of both 16S and 23S rRNA. 

Puromycin a nucleoside antibiotie (M, 472) from Sireptomyces alboniger. As a strudural aiialog of the 3 -terminal end of aminoacyl-tRNA (Hg.), P. inhibits protein biosynthesis on 70S and SOS rib mes. It replaces the 3 -terminus of aminoacyl-tRNA during the elongation phase of protein biosynthesis, and a pep-... 

Fig. 2. Consensus structure of the E. coli 70S ribosome and its subunits. A. B, C and D are different orientations of the large (SOS) subunit E, F, G and H are two orientations of the small (30S) subunit On the large subunit, E, M and P represent the nascent protein exit site, the membrane binding site, and the peptidyl transferase site, respectively. 23S 3 indicates the position of the 3 terminus of 23S rRNA. On the small subunit, IF-1,2,3 represents the probable location of initiation factors 1, 2 and 3. EF-Tu represents the binding site of the EF- Tu GTP aminoacyl-tRNA complex (see Protein biosynthesis). EF-G represents the binding site of elongation factor G (see Protein biosynthesis) near the interface area with the large subunit. 16S 3 and 16S 5 indicate the positions of the 3 and 5 termini of 16S rRNA. Numbers preceded by S and L represent ribosomal proteins of the small and large subunits, respectively, which have been mapped by electron microscopic visualization of subunit-antibody complexes. / is a diagrammatic representation of the whole ribosome, showing the probable location of mRNA and newly synthesized polypeptide, and the position and orientation of the ribosome with respect to the membrane of the endoplasmic reticulum during synthesis of secreted proteins.

Sites Studied. Interactions of tRNA with the ribosome can be broadly classified into two types, (a) Interactions with the peptidyltransferase center of the 50 S ribosomal subunit involving the 3 terminus of peptidyl-or aminoacyl-tRNA occupying, respectively, the donor (peptidyl) or acceptor (aminoacyl) site of the catalytic center, (b) Interactions likely to involve internal parts of the tRNA molecule and designed to align and stabilize the binding of cognate tRNAs to the ribosome. 

Puromycin has a structure closely analogous to the 3 terminus of a tyrosine-tRNA, and it inhibits protein synthesis by accepting, in place of an aminoacyl-tRNA, an incomplete polypeptide chain from ribosome-bound peptidyl-tRNA, thus prematurely terminating protein synthesis. We have found that irradiation at 2537 A (low-pressure Hg lamp) of solutions containing puromycin and ribosomes from E. coli leads to significant covalent incorporation of puromycin into the ribosome. For... 

Jonak, J., Rychlik, I., Smrt, J., and Holy, A., 1979, The binding site for the 3 -terminus of aminoacyl-tRNA in the molecule of elongation factor Tu from Escherichia coli, FEES Lett.,... 

During translation, the amino adds are attached to the 3 ends of their respective tRNAs. The aminoacyl-tRNAs are situated in the P and A sites of the ribosome as shown in Figure 1-4-8, Sotice that the peptide bond forms between the carboxyl group of the amino add (or growing peptide) in the P site and the amino group of the ne rt amino add in the A site. Proteins are syn-desized from the amino to the carboxyl terminus. 

Puromycin, made by the mold Streptomyces al-boniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the 3 end of an aminoacyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 27-31). However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. 

The structure of the precursor to the minor E. coli tyrosine tRNA, is shown in Fig. 28-10. This is encoded by the amber suppressor gene SupF (see Chapter 29). Transcription of its gene is initiated by GTP 43 bp upstream of the 5 end of the mature tRNA and usually terminates at a p-dependent signal 225 bp beyond the CCA terminus of the tRNA. An endonuclease cuts the transcript a few nucleotides beyond the CCA end. It is then trimmed to an 130-nucleotide piece still containing 2-3 extra nucleotides at the 3 end. This intermediate is cut by RNase P at the 5 end after which final trimming is done at the 3 end (Fig. 28-10).226 227... 

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