Nucleotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of<i>E. coli</i>tRNAs

Journal of Biological Chemistry

Björk²,

Fontecave³

et al. 2002

The product of the miaB gene, MiaB, from Escherichia coli participates in the methylthiolation of the adenosine 37 residue during modification of tRNAs that read codons beginning with uridine. A His-tagged version of MiaB has been overproduced and purified to homogeneity. Gel electrophoresis and size exclusion chromatography revealed that MiaB protein is a monomer. As isolated MiaB contains both iron and sulfide and an apoprotein form can chelate as much as 2.5-3 iron and 3-3.5 sulfur atoms per polypeptide chain. UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions, whereas [2Fe-2S] and [3Fe-4S] forms are generated under aerobic conditions. Preliminary site-directed mutagenesis studies suggest that Cys 157 , Cys 161 , and Cys 164 are involved in iron chelation and that the cluster is essential for activity. Together with the previously shown requirement of S-adenosylmethionine (AdoMet) for the methylthiolation reaction, the finding that MiaB is an iron-sulfur protein suggests that it belongs to a superfamily of enzymes that uses [Fe-S] centers and AdoMet to initiate radical catalysis. MiaB is the first and only tRNA modification enzyme known to contain an Fe-S cluster.

“…The cloned gene was completely sequenced to verify that the PCR had not introduced any errors. The pT 7 -7-derived plasmid containing the whole miaB gene was named pT 7 -miaB.…”

Section: Cloning Of the Miab Gene And Construction Of Plasmids Overexmentioning

confidence: 99%

“…Site-directed Mutagenesis-Mutagenesis was carried out on plasmid pT 7 -miaBH with QuikChange TM Site-Directed Mutagenesis kits from Stratagene according to the manufacturer's protocol. Mutations were confirmed by DNA sequencing.…”

Section: Cloning Of the Miab Gene And Construction Of Plasmids Overexmentioning

confidence: 99%

Enzymatic Modification of tRNAs

Journal of Biological Chemistry

Björk²,

Fontecave³

et al. 2002

“…This enzyme, the product of the miaB gene (Scheme 1), catalyzes a difficult C-H to C-S bond conversion during the thiomethylation of i 6 A-37 (N-6-isopentenyl adenosine) to ms 2 i 6 A-37 (2-methylthio-N-6-isopentenyl adenosine). This nucleoside is found at position 37, next to the anticodon at the 3Ј position in almost all eukaryotic and bacterial tRNAs that read codons beginning with uridine except tRNA I.V Ser (10). In Salmonella typhimurium, but not in Escherichia coli, for example, it is further modified by hydroxylation during an oxygen-dependent reaction catalyzed by the product of the miaE gene (Scheme 1) (11).…”

mentioning

confidence: 99%

tRNA-modifying MiaE protein fromSalmonella typhimuriumis a nonheme diiron monooxygenase

Mathevon

Proc. Natl. Acad. Sci. U.S.A.

Oddou

et al. 2007

MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mö ssbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.EPR spectroscopy ͉ Mö ssbauer spectroscopy ͉ nonheme dinuclear iron cluster ͉ tRNA modification enzyme

“…This is an oxidation reaction whose mechanism has yet to be investigated. The modified nucleoside ms 2 i 6 A is found at position 37, next to the anticodon on the 3Ј-position in almost all eukaryotic and bacterial tRNAs that read codons beginning with U except tRNA I,V Ser (3).…”

mentioning

confidence: 99%

MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA

Journal of Biological Chemistry

Douki

Fontecave

et al. 2004

157

180

The last biosynthetic step for 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyl-adenosine (i 6 A) precursor. In this work we have reconstituted in vitro the conversion of i 6 A to ms 2 i 6 A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i 6 A-containing RNA corresponding to the anticodon stem loop of tRNA Phe is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i 6 A. In this process, one molecule of AdoMet is converted to 5 -deoxyadenosine, probably through reductive cleavage and intermediate formation of a 5 -deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.In all organisms, the nucleosides of transfer RNA (tRNA) undergo different post-transcriptional modifications that are important for their biological activity (1). These modifications are part of the complex process of tRNA maturation and are introduced through the action of many different enzymes on the polynucleotide transcripts (2). Although modified bases are found at different positions in tRNA, the anticodon loop is the most heavily modified region of the molecule and contains the greatest variety of modified nucleosides. To date, a total of 86 structurally distinguishable modified nucleosides in tRNA from many diverse organisms of the three major phylogenetic domains of life have been identified (medstat.med.utah.edu/ RNAmods/). Among these modified nucleosides, the so-called thionucleosides containing a sulfur atom have been the subject of particular attention in the recent years.In Escherichia coli four different thiolated nucleosides have been characterized; these are 4-thiouridine (s 4 U), 1 2-thiocytidine (s 2 C), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), and 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A). If the synthesis of thiopyrimidines involves a non-redox substitution reaction of an oxygen atom by sulfur, the reaction leading to the synthesis of 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A) consists of a chemically highly challenging aromatic C-H to C-S bond conversion. This is an oxidation reaction whose mechanism has yet to be investigated. The modified nucleoside ms 2 i 6 A is found at position 37, next to the anticodon on the 3Ј-position in almost all eukaryotic and bacterial tRNAs that read codons beginning with U except tRNA I,V Ser (3).Initial studies on ms 2 i 6 A biosynthesis in E. coli and Salmonella typhimurium established a requirement for at least two enzymes (4). The first step consists in the addition of the isopentenyl group to the N 6 nitrogen of adenosine. This reaction is catalyzed by the well characterized tRNA-isopentenylpyrophosphate transferase enzyme, encoded by the miaA gene (5...