tRNA Trp Research Articles

Mitochondrial genome sequences of landsnails Aegista diversifamilia and Dolicheulota formosensis (Gastropoda: Pulmonata: Stylommatophora)

The first mitochondrial genome sequences of Aegista and Dolicheulota belonging to Bradybaenidae are described in this report. Mitogenomic sequences were generated from Illumina paired-end sequencing. The complete mitogenome of Aegista diversifamilia was 14,039 bp in length and nearly complete mitogenome of Dolicheulota formosensis was 14,237 bp. Both mitogenomes consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. Most genes were overlapped with neighboring genes that the overlapping regions ranged from 2 to 64 bp in A. diversifamilia and from 1 to 45 bp in D. formosensis. Novel gene arrangement, tRNA–Tyr–ND3–tRNA–Trp, was identified in A. diversifamilia, whereas D. formosensis showed identical gene order to other Bradybaenidae mitogenomes. Maximum likelihood phylogenetic tree suggested Aegista as a sister clade to Euhadra and Dolicheulota. Bradybaenidae is monophyly sister clade to Camaenidae.

Abstract 4323: Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients

Abstract Introduction: Mitochondrial dysfunction is related to the genesis of many types of cancers, including breast cancer. Mitochondrial tRNA genes perform several functions including processing and translation in which are essential components of mitochondrial protein synthesis. Until now only few somatic mitochondrial tRNA mutations have been reported in cancer cells. The aim of this study was to find out the relationship between mitochondrial DNA mutations and breast cancer. Patients and Methods: In current this study, 22 mitochondrial tRNAs genes in 30 tumoral tissues and adjacent non-tumoral tissues of patients with breast cancer and 100 blood samples of healthy controls were investigate using PCR-Sequencing methods. Results and conclusions: From 30 breast cancer cases, twelve genetic variations were found in 22 tRNA MtDNA. Six of twelve (50%) were novel and remaining was polymorphism. Among new variants, which incloding C1631A (tRNA Val), T1633A (tRNA Val), A5565G (tRNA Trp), G5849A (tRNA Tyr) were at hetroplasmic state and remaining which including C12187 (tRNA His) and A15948G (tRNA Thr) were at homoplasmic. Among variants, A12308G, a polymorphic mutation in V-loop (tRNALeu(CUN)), was found in 23% breast tumoral tissues (7 of 30) and 3%in healthy blood controls (3 of 100), indicating a statistically significant association of the A12308G alteration between tumoral tissues and controls(p < 0.05).Our finding indicate that , the A12308G alteration may increase the risk of breast cancer through its impact on the tRNA structure result in mitochondrial dysfunction. Therefore, it may consider as a pathogenic predisposition factor for the breast cancer risk. Note: This abstract was not presented at the meeting. Citation Format: Sara Eslamizadeh, Massoud Ghaffarpour, Mohammad Arabzadeh, Massoud Houshmand. Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2014-4323

Chloro- and Bromo-Tryptophan Analogs as Phosphorescent Probes in Proteins

Tryptophan phosphorescence spectroscopy is one of the most sensitive methodologies around to study the local protein structure. For proteins in buffer, the phosphorescence lifetime (τp) directly reports about the local flexibility of the site with τp ranging from (sub) milliseconds for Trp at a solvent exposed position to several seconds if the Trp side chain is embedded in a rigid protein core. Because of the long intrinsic τp of Trp, 6.5 s, the method is extremely sensitive for quenchers e.g. oxygen and its concentration needs to be reduced to a level of several nM. This extreme sensitivity for quenchers has prevented widespread use of Trp phosphorescence in molecular biology. Substitution of the indole ring with a heavy atom is expected to significantly reduce the intrinsic τp, and thus the sensitivity for quenchers. We measured intrinsic τp values of 400 ms for 6-chloroindole, 13 ms for 6- bromoindole and 38 ms for 5-bromoindole, demonstrating the potential of these Trp analogs as phosphorescence probes in proteins. A straightforward approach to produce proteins labeled with these analogs was not available. We developed before a Trp auxotrophic strain of Lactococcis lactus able to incorporate Trp analogs in proteins which E. coli Trp auxotrophs do not translate. However, this L. lactus strain was found not able to translate bromo- or cloro-substituted Trp analogs. To overcome this, the aminoacyl t-RNA Trp synthetase enzyme from L. lactus was cloned and overexpressed in L. lactus together with a single-Trp containing target protein. This approach resulted in the efficient incorporating (87-91%) of the halogenated Trp analogs and a high protein yield (25-50 mg/L culture). Details of the methodology are presented as well as the spectroscopic characterization of the chloro- and bromo-Trp containing proteins.

The Nop5–L7A–fibrillarin RNP complex and a novel box C/D containing sRNA of Halobacterium salinarum NRC-1

RNA 2′O-methylation is a frequent modification of rRNA and tRNA and supposed to influence RNA folding and stability. Ribonucleoprotein (RNP) complexes, containing the proteins Nop5, L7A, fibrillarin, and a box C/D sRNA, are guided for 2′O-methylation by interactions of their RNA component with their target RNA. In vitro complex assembly was analyzed for several thermophilic Archaea but in vivo studies are rare, even unavailable for halophilic Archaea. To analyze the putative box C/D RNP complex in the extremely halophilic Halobacterium salinarum NRC-1 we performed pull-down analysis and identified the proteins Nop5, L7A, and fibrillarin and the tRNA Trp intron, as a typical box C/D sRNA of this RNP complex in vivo. We show for the first time a ribonucleolytic activity of the purified RNP complex proteins, as well as for the RNP complex containing pull-down fractions. Furthermore, we identified a novel RNA (OE4630R-3′sRNA) as part of the complex, containing the typical boxes C/D and C′/D′ sequence motifs and being twice as abundant as the tRNA Trp intron.

Plant mitochondrial RNA editing: The Strasbourg chapter

For 8 years, it was not understood why certain genes of plant mitochondria contain CGG (arginine) codons at positions where tryptophan codons (UGG) are present in the corresponding genes of nonplant species. Identification and sequencing of a tRNA(Trp) gene showed that it is not able to decode the CGG codon. Analysis of different discrepancies in the sequences of plant mitochondrial proteins prompted us to determine directly the corresponding RNA sequences. These experiments showed that plant mitochondrial transcripts are subject to RNA editing that changes C into U, resulting in a better phylogenetic conservation of protein sequences [Gualberto et al. (1989) Nature 341, 660-662].

Species-specific differences in the regulation of the aminoacylation activity of mammalian tryptophanyl-tRNA synthetases

Tryptophanyl-tRNA synthetases (TrpRSs) catalyze the aminoacylation of tRNA Trp. Previously, I demonstrated that Zn 2+-depleted human TrpRS is enzymatically inactive and that binding of Zn 2+ or heme to human TrpRS stimulates its aminoacylation activity. In the present study, bovine and mouse TrpRSs were found to be constitutively active regardless of the presence of Zn 2+ or ferriprotoporphyrin IX chloride. Mutagenesis experiments demonstrated that the human H130R mutant is constitutively active and that the bovine R135H, E438A double mutant binds with Zn 2+ or heme to enhance its aminoacylation activity as does human wild-type TrpRS. These results provide the first evidence of species-specific regulation of TrpRS activity.

Two new mutations in the MT-TW gene leading to the disruption of the secondary structure of the tRNA Trp in patients with Leigh syndrome

Leigh syndrome is a progressive neurodegenerative disorder occurring in infancy and childhood characterized in most cases by a psychomotor retardation, optic atrophy, ataxia, dystonia, failure to thrive, seizures and respiratory failure. In this study, we performed a systematic sequence analysis of mitochondrial genes associated with LS in Tunisian patients. We sequenced the encoded complex I units: ND2, ND3, ND4, ND5 and ND6 genes and the mitochondrial ATPase 6, tRNA Val, tRNA Leu(UUR), tRNA Trp and tRNA Lys genes in 10 unrelated patients with Leigh syndrome. We revealed the presence of 34 reported polymorphisms, nine novel nucleotide variants and two new mutations (T5523G and A5559G) in the tested patients. These two mutations were localized in two conserved regions of the tRNA Trp and affect, respectively, the D-stem and the T-stem of the mitochondrial tRNA leading to a disruption of the secondary structure of this tRNA. SSP-PCR analysis showed that the T5523G and A5559G mutations were present with respective heteroplasmic rates of 66% and 43 %. We report here the first mutational screening of mitochondrial mutations in Tunisian patients with Leigh syndrome which described two novel mutations associated with this disorder.

The complete mitochondrial genome of Macaca thibetana and a novel nuclear mitochondrial pseudogene

The complete mitochondrial genome of Macaca thibetana was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequencing methods. It is 16,540 bp and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region. Most codon usage followed the typical pattern of vertebrates. Two rare start codons were found, in which GTG initiated the NADH dehydrogenase subunit 1 (ND1) gene and the ATP8 gene, and ATT initiated the ND2 gene. A new mitochondrial DNA-like sequence (2003 bp) in the nuclear genome of M. thibetana was found. It matched with the 3′ end of the ND1 gene, the tRNA Ile-tRNA Gln-tRNA Met genes, the ND2 gene, and the 5′ end of the tRNA Trp gene. Sequence divergence between the nuclear pseudogene and the mitochondrial homologue suggested that the translocation of this mtDNA fragment into the nuclear genome occurred 3.16 ∼ 3.48 million years ago (MYA). Molecular phylogenetic analysis of 16 Cercopithecidae species was performed using sequences from 12 concatenated heavy-strand encoded protein coding genes. The results provided more evidence to support previous morphological and chromosomal studies on Cercopithecidae.

The Presence of a C − 1 /G + 73 Pair in a tRNA Precursor Influences Processing and Expression In Vivo

To understand whether 5′ and 3′ structural elements of the region corresponding to the mature tRNA affect the expression of the tRNA, we examined several bacterial genomes for tRNA genes where the expression might be potentially affected by structural elements located outside of the mature tRNA. In Pseudomonas aeruginosa, our analysis suggested that the tRNA Trp is transcribed together with a putative stem–loop structure followed by a uridine tract immediately downstream of the tRNA region. This structural element, resembling a Rho-independent transcription terminator, might therefore influence the expression and processing of tRNA Trp. Moreover, the secondary structure suggested that the discriminator base in the tRNA Trp precursor can pair with either the C at position − 1, the 3′ terminal residue in the 5′ leader, or the C immediately 5′ of the uridine tract of the putative Rho-independent transcription terminator. Here, we present in vivo data demonstrating the importance of residue − 1 and the positioning of the putative transcription terminator for the expression of correctly 5′ processed P. aeruginosa tRNA Trp in Escherichia coli. Interestingly, we also detected a difference in the appearance of correctly 5′ processed P. aeruginosa tRNA Trp in E. coli compared to the situation in P. aeruginosa.

The complete mitochondrial genomes of needle corals, Seriatopora spp. (Scleractinia: Pocilloporidae): An idiosyncratic atp8, duplicated trnW gene, and hypervariable regions used to determine species phylogenies and recently diverged populations

Complete DNA sequences were determined for the mitochondrial (mt) genomes of the needle corals, Seriatopora caliendrum (17,011 bp) and S. hystrix (17,060 bp). Gene arrangement of the Seriatopora mt genomes is similar to the 14 currently published scleractinian mitogenomes with three unusual features, including an idiosyncratic atp8, a duplicated trnW ( tRNA TRP ), and a putative control region located between atp6 and nad4. A tp8, located between duplicate trnW genes, showed relatively low amino acid similarity (25.6–34.6%) with those of published scleractinian corals. A reverse-transcription polymerase chain reaction confirmed the transcription of this novel atp8 gene in Seriatopora. A duplicated trnW was detected in the region close to the cox1 gene and shares the highly conserved primary and secondary structure of its original counterpart. The intergenic spacer between atp6 and nad4, which contains several distinct repeated elements, is being designated as the putative control region in the Seriatopora mt genomes. Evaluation of the molecular evolution of several protein-coding genes and intergenic spacers showed 3- to 4-fold higher divergence rates among populations or between species than those published for scleractinian mt genomes. This study not only successfully revealed the phylogenies of S. hystrix and S. caliendrum from the West Pacific Ocean by mtDNA, but also highlighted the potential utilities of mt hypervariable regions in phylogenetic construction below the species level for Seriatopora.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

Comparison of tryptophan biosynthetic operon regulation in different Gram-positive bacterial species

The tryptophan biosynthetic operon has been widely used as a model system for studying transcription regulation. In Bacillus subtilis, the trp operon is primarily regulated by a tryptophan-activated RNA-binding protein, TRAP. Here we show that in many other Gram-positive species the trp operon is regulated differently, by tRNA(Trp) sensing by the RNA-based T-box mechanism, with T-boxes arranged in tandem. Our analyses reveal an apparent relationship between trp operon organization and the specific regulatory mechanism(s) used.

The G490E mutation in reverse transcriptase does not impact tRNA primer selection by HIV-1 with altered PBS and A-loop

The initiation of HIV-1 reverse transcription utilizes a cellular tRNA Lys,3 as a primer. The 3′ terminal 18-nucleotides of the cellular tRNA Lys,3 are complementary to a region on the viral genome, designated as the primer binding site (PBS). Previous studies have shown that forcing HIV-1 to utilize alternative tRNA primers through alteration of the PBS results in viruses that revert to utilize tRNA Lys,3 following in vitro replication. In some instances though, HIV-1 has been shown to select alternative tRNAs for initiation of reverse transcription if additional mutations upstream in the U5 region (A-loop) were made to be complementary to these alternative tRNAs. Recently, an HIV-1 has been described in which the U5 region distinct from the A-loop, designated as the primer activation site (PAS), was mutated in conjunction with the PBS to force the virus to use tRNA Lys1,2 as a primer. An additional mutation in reverse transcriptase (RT), G490E, was found to facilitate the forced use of tRNA Lys1,2 as the primer. In the current study, we have investigated the impact of the G490E mutation in RT on the selection and use of alternative primers by HIV-1. Viruses were first constructed in which the PBS and A-loop region were made complementary to tRNA Trp. Previous studies from our laboratory have shown that these viruses are unstable and mutate to select tRNA Met or tRNA Lys1,2. Analysis of the replication of viruses with the U5 and PBS complementary to tRNA Trp with or without the G490E mutation revealed no significant differences with respect to infectivity and viral growth in SupT1 or peripheral blood mononuclear cells (PBMC). In addition, the presence of the G490E mutation did not influence the capacity of this virus to revert to utilize tRNA Met as the primer for initiation of reverse transcription. In a previous study, we have described an HIV-1 that has been forced to utilize tRNA Lys1,2 through mutations in the A-loop and PBS. The G490E RT mutation in this virus did not impact on the virus infectivity or growth in SupT1 or PBMC. We did not find a significant fitness advantage to viruses in which the A-loop and PBS were made complementary to tRNA Lys1,2 that also contained the G490E mutation in RT. The results of these studies then establish that HIV-1 can be forced to use alternative primers through mutations in the U5 (PAS or A-loop) for certain tRNAs. Furthermore, for mutations in the A-loop and PBS, the RT does not play an important role in dictating the selection of the alternative primers to be used for initiation of reverse transcription.

A molecular assessment of phylogenetic relationships and lineage accumulation rates within the family Salamandridae (Amphibia, Caudata)

We examine phylogenetic relationships among salamanders of the family Salamandridae using approximately 2700 bases of new mtDNA sequence data (the tRNA Leu, ND1, tRNA Ile, tRNA Gln, tRNA Met, ND2, tRNA Trp, tRNA Ala, tRNA Asn, tRNA Cys, tRNA Tyr, and COI genes and the origin for light-strand replication) collected from 96 individuals representing 61 of the 66 recognized salamandrid species and outgroups. Phylogenetic analyses using maximum parsimony and Bayesian analysis are performed on the new data alone and combined with previously reported sequences from other parts of the mitochondrial genome. The basal phylogenetic split is a polytomy of lineages ancestral to (1) the Italian newt Salamandrina terdigitata, (2) a strongly supported clade comprising the “true” salamanders (genera Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), and (3) a strongly supported clade comprising all newts except S. terdigitata. Strongly supported clades within the true salamanders include monophyly of each genus and grouping Chioglossa and Mertensiella as the sister taxon to a clade comprising Lyciasalamandra and Salamandra. Among newts, genera Echinotriton, Pleurodeles, and Tylototriton form a strongly supported clade whose sister taxon comprises the genera Calotriton, Cynops, Euproctus, Neurergus, Notophthalmus, Pachytriton, Paramesotriton, Taricha, and Triturus. Our results strongly support monophyly of all polytypic newt genera except Paramesotriton and Triturus, which appear paraphyletic, and Calotriton, for which only one of the two species is sampled. Other well-supported clades within newts include (1) Asian genera Cynops, Pachytriton, and Paramesotriton, (2) North American genera Notophthalmus and Taricha, (3) the Triturus vulgaris species group, and (4) the Triturus cristatus species group; some additional groupings appear strong in Bayesian but not parsimony analyses. Rates of lineage accumulation through time are evaluated using this nearly comprehensive sampling of salamandrid species-level lineages. Rate of lineage accumulation appears constant throughout salamandrid evolutionary history with no obvious fluctuations associated with origins of morphological or ecological novelties.

Molecular phylogenetics of theAnolis onca series: a case history in retrograde evolution revisited

Anoles of the Anolis onca series represent a dramatic case of retrograde evolution, exhibiting great reduction (A. annectens) and loss (A. onca) of the subdigital pads considered a key innovation for the evolutionary radiation of anoles in arboreal environments. We present a molecular phylogenetic analysis of these anoles and their closest known relatives (A. auratus, A. lineatus, A. meridionalis, and A. nitens) using new mitochondrial DNA sequence data from the ND2 gene, five tRNA genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr)), the origin of light-strand replication, and a portion of the CO1 gene (1,446 aligned base positions, 612 parsimony informative). Our results confirm monophyly of the A. onca series and suggest an evolutionary separation of approximately 10 million years between A. annectens and A. onca. Evolution of subdigital structure in this series illustrates ectopic expression of developmental programs that replace flexible subdigital lamellae of the toepad with rigid, keeled scales resembling dorsal digital scales. Our phylogenetic results indicate that narrowing of the toepad in A. auratus evolved separately from toepad reduction in the A. onca series. Expansion of the subdigital lamellae along the phalanges in A. auratus appears to compensate constriction of lamellae by digital narrowing, maintaining greater climbing capability in this species. Toepad evolution in the lineage ancestral to A. auratus features changes of the same developmental modules as the A. onca series but in the opposite direction. Large molecular distances between geographic populations of A. auratus indicate that its derived toepad structure is at least 9 million years old.

The complete set of tRNA species in Nanoarchaeum equitans

The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requiring the assembly of seperate 5′ and 3′ tRNA halves [Randau, L., Münch, R., Hohn, M.J., Jahn, D. and Söll, D. (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves. Nature 433, 537–541]. Biochemical evidence was missing for one of the computationally-predicted, joined tRNAs designated as tRNA Trp. Our RT-PCR and sequencing results identify this tRNA as tRNA Lys (CUU) joined at the alternative position between bases 30 and 31. We show that the intron-containing tRNA Trp was misidentified in the initial Nanoarchaeum equitans genome annotation [E. Waters et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100, 12984–12988]. Along with a previously unidentified joined tRNA Gln (UUG), Nanoarchaeum equitans exhibits 44 tRNAs and is enabled to read all 61 sense codons. Features unique to this set of tRNA molecules are discussed.

Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum

To understand the decoding property of nematode mitochondrial tRNAs with unusual secondary structures, post-transcriptional modifications at wobble positions of Ascaris suum mitochondrial tRNAs corresponding to two-codon families ending with a purine were analyzed. 5-Carboxymethylaminomethyluridine (cmnm 5U) was identified at the wobble positions of tRNA Lys, tRNA Glu and tRNA Gln, while 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5s 2U) was present in tRNA UAA Leu and tRNA Trp . In most bacterial and mitochondrial tRNAs, the 2-thiouridine derivative is present in tRNAs for Lys, Glu and Gln. These is no report that cmnm 5s 2U is used in tRNA UAA Leu and tRNA Trp . The unusual usage of wobble modifications might assist decoding of nematode mitochondrial mRNAs.

Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNA Trp. In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNA Glu. Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TψC loop of tRNA Leu(UUR). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92–96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.

Interaction of the trp RNA-binding Attenuation Protein (TRAP) with Anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5′-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA Trp induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP–AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of ∼3×10 14 M −3. About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP–AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.