Structure and Organization of Genes

Reeve, John N.

doi:10.1007/978-1-4615-2391-8_13

Cited by 18 publications

(23 citation statements)

References 150 publications

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“…2 and 4). Transcription of genes from the Archaea typically initiates 22 to 27 bp downstream of a box A sequence [consensus, 5Ј-TTA(T/A)ATA-3Ј] at a purine-pyrimidine dinucleotide, usually in a box B sequence (consensus, 5Ј-ATGC-3Ј) (33). One of the 5Ј ends (nucleotide Ϫ189) mapped to a purine-pyrimidine dinucleotide which was located 20 bp downstream of a 6-bp sequence (5Ј-TATATT-3Ј) with identity to the consensus box A (Fig.…”

Section: Vol 178 1996mentioning

confidence: 99%

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila

Maupin-Furlow

Ferry

1996

J Bacteriol

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The cdhABC genes encoding the respective ␣, , and ␤ subunits of the five-subunit (␣, ␤, ␥, ␦, and ) CO dehydrogenase/acetyl-coenzyme synthase (CODH/ACS) complex from Methanosarcina thermophila were cloned and sequenced. Northern (RNA) blot analyses indicated that the cdh genes encoding the five subunits and an open reading frame (ORF1) with unknown function are cotranscribed during growth on acetate. Northern blot and primer extension analyses suggested that mRNA processing and multiple promoters may be involved in cdh transcript synthesis. The putative CdhA (␣ subunit) and CdhB ( subunit) proteins each have 40% identity to CdhA and CdhB of the CODH/ACS complex from Methanosaeta soehngenii. The cdhC gene encodes the ␤ subunit (CdhC) of the CODH/ACS complex from M. thermophila. The N-terminal 397 amino acids of CdhC are 42% identical to the C-terminal half of the ␣ subunit of CODH/ACS from the acetogenic anaerobe Clostridium thermoaceticum. Sequence analysis suggested potential structures and functions for the previously uncharacterized ␤ subunit from M. thermophila. The deduced protein sequence of ORF1, located between the cdhC and cdhD genes, has 29% identity to NifH2 from Methanobacterium ivanovii.The five-subunit (␣, ␤, ␥, ␦, and ε) CO dehydrogenase/ acetyl-coenzyme A (CoA) synthase (CODH/ACS) complex is central to the pathway for the fermentation of acetate to CH 4 and CO 2 in the methanoarchaeon Methanosarcina thermophila (reviewed in reference 8), where it functions to cleave the C-C and C-S bonds in the acetyl moiety of acetyl-CoA, oxidize the carbonyl group to CO 2 , and transfer the methyl group to tetrahydrosarcinapterin (THSPt). The complex also catalyzes CO oxidation-CO 2 reduction (CO dehydrogenase activity) and synthesis of acetyl-CoA with CO, a methyl group, and CoA; however, the synthesis activity is not important in the fermentation pathway. A membrane-bound methyltransferase transfers the methyl group from methyl-THSPt to CoM. The methyl-CoM is reductively demethylated to CH 4 with electrons that originate from oxidation of the carbonyl group of acetyl-CoA to CO 2 by the CODH/ACS enzyme complex. Detergent treatment resolves the five-subunit CODH/ACS complex into two enzyme components: the nickel-iron-sulfur (␣ε) component contains the ␣ and ε subunits, and the corrinoid-iron-sulfur (␥␦) component contains the ␥ and ␦ subunits of the complex (1). The ␤ subunit is also resolved, but it is unstable and has not been characterized. The resolved ␣ε component has CO dehydrogenase activity and, therefore, is proposed to oxidize the carbonyl group of acetyl-CoA to CO 2 (1). Electron paramagnetic resonance (EPR) spectroscopy identifies three metal clusters (A, B, and C) in the ␣ε component (21) which have EPR spectroscopic properties indistinguishable from those of clusters A, B, and C in the well-characterized ␣ 2 ␤ 2 CODH/ACS from Clostridium thermoaceticum (reviewed in reference 29). The CODH/ACS from C. thermoaceticum functions to synthesize acetyl-CoA from CoA, CO, and a methyl group donated by a co...

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Section: Vol 178 1996mentioning

confidence: 99%

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila

Maupin-Furlow

Ferry

1996

J Bacteriol

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“…The biochemistry of carbon dioxide fixation from H 2 and CO 2 in this organism is now known in great detail (8), and several of the genes involved have been cloned and sequenced (14,22). Growth requirements of methanogens have been extensively investigated and include several metals, most notably iron, nickel, cobalt, selenium, and molybdenum or tungsten (4,15).…”

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Purification and structural characterization of a flavoprotein induced by iron limitation in Methanobacterium thermoautotrophicum Marburg

1995

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“…Two triplets, an ATT and a TTG, which are known as unusual start codons in eubacteria (9) and archaea (18), are located in frame upstream from the originally proposed ATG start codon.…”

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“…In bacteria, apart from the commonly used ATG, the triplets GTG, ATT, and TTG serve as initiation codons (9). In methanogens, genes initiated with TTG or, more frequently, with GTG have been identified (18). The occurrence of the rare start codon TTG in methanogenic archaea is summarized in Table 1.…”

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“…Furthermore, upstream of virtually every methanogen open reading frame there is a sequence which shows homology to a Shine-Dalgarno sequence. The methanogen Shine-Dalgarno sequences and the other elements determining the translation initiation region, including the ATG start codon, seem to function in E. coli so that methanogen genes (under the control of an E. coli promoter) can be expressed correctly in E. coli (18). To see if this holds true when TTG, not ATG, is used as a start codon, we have expressed the gene for MvaS7 via its own translation initiation region in E. coli.…”

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TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii

et al. 1995

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The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.The gene for ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is part of a transcriptional unit which corresponds to the eubacterial streptomycin operon. Apart from the presence of two additional open reading frames and the gene for MvaS10, the organization of the streptomycin operon of M. vannielii (open reading frame 1 [ORF1]-ORF2-MvaS12-MvaS7-EF2-EF1␣-MvaS10) is identical to that of Escherichia coli. In E. coli the S10 gene is the first gene in the S10 operon. On the basis of the nucleotide sequence, MvaS7 was described as a protein of 146 amino acids (16). More recent homology studies of ribosomal S7 proteins from the archaea Haloarcula marismortui, Halobacterium morrhuae, and M. vannielii suggested that MvaS7 should be more than 40 amino acids longer than previously suggested (14).Analysis of the nucleotide sequence preceding the ATG start codon proposed by Lechner et al. (16) revealed an amino acid sequence which shows a high degree of homology to the sequences of the other archaeal S7 proteins. Two triplets, an ATT and a TTG, which are known as unusual start codons in eubacteria (9) and archaea (18), are located in frame upstream from the originally proposed ATG start codon.In this communication we report that MvaS7 is a protein of 188 amino acids, i.e., 42 amino acids longer than originally proposed. Furthermore, we demonstrate that TTG is used as a start codon and that the methanogenic translation initiation region containing TTG as a start codon is recognized in E. coli.Cloning and expression in E. coli of three size variants of the gene encoding MvaS7. Three codons, namely, ATT (Fig. 1, position 2172), TTG (Fig. 1, position 2190), and ATG (Fig. 1, position 2317), are potential candidates for the start codon of the MvaS7 gene. To express the three possible MvaS7 variants in E. coli BL21(DE3) (21), we have chosen vector pET 11a (6), since the expression of cloned genes can be strictly regulated. If MvaS7, the homolog of the autoregulatory protein S7 of the streptomycin operon in E. coli (4), functioned in E. coli, we could not exclude the possibility that the basal expression level of MvaS7 in the uninduced state might affect growth of the E. coli host. To clone the three MvaS7 gene variants, we have used the oligonucleotides shown in Fig. 1 as primers to make PCR products containing an NdeI site and a BamHI site. The PCR protocol and cloning of the PCR products into pET 11a via pUC 18 are detailed in reference 10. To allow cloning and efficient expression, the rare start codons ATT and TTG were replaced by an ATG. The constructs were designated pMvaS7 Identification of MvaS7. Our aim was to identify MvaS7 in the two-dimensional protein patt...

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Structure and Organization of Genes

Cited by 18 publications

References 150 publications

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila

Purification and structural characterization of a flavoprotein induced by iron limitation in Methanobacterium thermoautotrophicum Marburg

TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii

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