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Based on the genetic context, two trp gene clusters were identified which contain the enzymes on the branch leading from chorismate to tryptophan. As seen in figure #82A, the first trp cluster consists of the overlapping ORFs OE1469F, OE1470F and OE1471F, followed by a 25bp sequence which connects ORF OE1472F to the cluster. The second trp cluster seen in figure #82B, trpD1FE1G1, consists of four overlapping

ORFs. Transcription of both clusters was strongly induced by the absence of AroAAs in the growth medium (figure #82).

Each of the trp clusters can be described as operons, as each possesses a common promoter region. In the first cluster (figure #82A), a putative TATA box promoter element was identified at position -33 from the starting codon of ORF OE1469F, while the 25bp sequence between ORF OE1471F and OE1472F, does not contain any TATA box sequence. The second trp cluster (figure #82B), contained a putative TATA box promoter element sequence at position -49. Both TATA box motifs were consistent with the conserved 8 bp promoter sequence element (TTTAWATR, with W=A or T, R=A or G) found in most Archaea (Gregor and Pfeifer, 2005). In a study by Koide et al., (Koide et al., 2009; Price et al., 2005), both of the trp clusters were identified as operons in H. salinarum sp. NCR-1, based on genomic-specific distance models and transcription start and end points of annotated operons by Koide et al,.

Figure # 82: AroAAs biosynthesis genes that display operon-like organization in H.

salinarum. A and B: the two trp operons, C: the tyrA operon, D-aroK-pheA1 operon. The fold regulations as found in the DNA microarray experiment are indicated below the ORFs. a_-

fold regulation was calculated from RT-qPCR experiment using two biological repeats. *- represents a 4bp overlapping region, **-represents a 10bp overlapping region, ***-represents a 66bp overlapping region. The putative TATA box promoter motifs are indicted. CHY- conserved hypothetical protein.

In H. salinarum, H. salinarum sp NCR-1, H. marismortui, H. walsbyi and N. pharaonis, the genes specifying the seven enzymatic reactions leading to the

formation of tryptophan from chorismate are organized in a similar arrangement, meaning split into two separated operons (figure #83). This is different to the arrangement of E. coli and methanococcus. For example, Methanobacterium thermoautotrophicum, possesses all seven trp genes adjacent to each other in the order trpEGCFBAD (Gast et al., 1994; Meile et al., 1991), as do some other Archaea (figure #5, (Xie et al., 2003)). Surprisingly, the trp operon in M. maripaludis is organized in the same way as in E. coli, trpEDCBA but, in contrast to E. coli, it does not contain a 14 amino acid leader peptide immediately preceding trpE (figure #85). Although it is believed that essential metabolic pathways have been likely transferred horizontally between Archaea and Bacteria, the histidine operon in E. coli, hisGDCNBHAFI, is another example were genes clustered in operon, were either scattered thorough the halobacterials genome (i.e. H. salinarum, N. pharaonis, and H. walsbyi), or partially clustered (i.e. P. furiosus) (Fondi et al., 2009).

Figure # 83

: trp operons in different archaea. trpG1E1FD1 (left panel) and trpCBA-X (right

panel) in different archaea. Halsp- Halobacterium sp. NRC-1, Halma-Haloarcula

marismortui ATCC 43049, Halwa- Haloquadratum walsbyi DSM 16790, Natph-

Natronomonas pharaonis DSM2160, Metja- Methanocaldococcus jannascii DSM 2661.

Modified from

Based on the microarray results, the overlapping gene pairs, tyrA-CHY, and aroK-

pheA1 are also likely to form operons, as depicted in figure #82C, D, respectively. Both clusters were identified as operons in H. salinarum sp NCR-1 based on transcription start and end points (Koide et al., 2009; Price et al., 2005). The tyrA- CHY operon is composed of ORF OE2770F (tyrA) and a conserved hypothetical protein (CHY, ORF OE2772F). Both ORFs are regulated according to the DNA microarray results obtained from synthetic medium lacking AroAAs, indicating that the CHY is a utilized ORF and potentially significant for biosynthesis or regulation of AroAAs. One the basis of its position, it is possible that this CHY could catalyse the

formation of tyrosine from 4-hydroxyphenylpyruvate, which is catalyzed by tyrB in E. coli (EC 2.6.1.57 or EC 2.6.1.5, aromatic-amino-acid transaminase, and tyrosine transaminase, respectively). No candidates for tyrB have been identified in haloarchaea by sequence similarity to the E. coli gene, suggesting that ORF OE2772F might be either a new member of transaminase family (although no conserved motifs were found), or has some other, as yet unknown, role in the AroAAs pathway. In E. coli, tyrA clusters with aroF which catalyses the first step in aromatic amino acid biosynthesis from the precursors PEP and E-4-P.

As seen in figure #82D, transcription of the gene cluster aroK-pheA1, was regulated by AroAAs and was found to be conserved in haloarchaea but not in M. maripaludis, M. jannaschii or E. coli (figure #84).

Figure # 84

: tyrA and aroK-pheA1 operons in different archaea. Halsp- Halobacterium sp. NRC-1, Halma-Haloarcula marismortui ATCC 43049, Halwa- Haloquadratum walsbyi

DSM 16790, Natph- Natronomonas pharaonis DSM2160, Metja- Methanocaldococcus

jannaschii DSM 2661, Esc05-E.coli K12. Modified from

(Lemoine et al., 2008).

As seen in figure #83 and 84, the trpG1E1FD1, trpCBA-OE1472F, tyrA-CHY and

aroK-pheA1 operons are conserved in Halobacterium sp. NRC-1, H. salinarum,

Haloarcula marismortui, Haloquadratum walsbyi, and Natronomonas pharaonis.

Differences are seen in M. jannaschii, in which the operons are missing or partly missing.

An arrangement of genes in operons confers both advantages and disadvantages. The most obvious advantage is that genes with similar function are transcribed together. The greatest disadvantage is that, unless some further level of regulation exist (differences in the amount of mRNA or its stability, the strength of ribosomal binding sites, and so on), the amount of polypeptides from those genes will be the same even though resultant enzymes may have different catalytic rates (Glansdorff,

1999). The enzymes with slower rates will be the limiting factor in the pathway. As a result, when genes are transcribed together, an excess of some enzymes is likely to occur. However, the amount of the mRNA and polypeptide synthesis is only one aspect of the control of the tryptophan pathway. In E. coli, besides these, there are two other levels of control that affect the amount of tryptophan synthesis within the cell. The first is feed-back inhibition which influence the activity of the first reaction (figure #86), and thereby the amount of metabolites flowing through the pathway. The second is the formation of multi-enzyme complexes that greatly increases the catalytic efficiency of the various reactions. In complexes, the product of one reaction can be used directly by the next enzyme because the concentration of the substrate in the vicinity of the second enzyme is much higher than would occur were the two enzymes separate. Examples of such complexes are trpE-trpG(D) and trpA-trpB (Ikeda, 2006). The transcription of trp operon and pheA in E.coli are regulated by attenuation, in response to changing intracellular concentrations of trp and phe, in the cell (Yanofsky, 1981). A leader peptide contains “control codons” that specify the amino acid end product of the enzymes encoded in the operon. When the supply of amino acid and thus of cognate aminoacyl-tRNA is deficient, the ribosome stalls at the control codons, the leader RNA forms a preemptor 2:3 hairpin, and transcription starts (figure #85C). If the aminoacyl-tRNA is abundant, the ribosome stops at the end of the leader peptide, thereby forming the prohibiting 3:4 hairpin, and transcription of the remainder of the operon does not occur (attenuation, figure #85B). In E. coli, the leader peptide of the trp operon contains 2 trp codons, while 7 phe codons precede the Phe ORF (Gollnick and Babitzke, 2002). It seems that transcriptions of the trp operons in H. salinarum are not regulated by attenuation, as no leader peptide was identified, and moreover, no trp codons were identified upstream to the first ORF in the operons.

Figure # 85: Regulation by transcription attenuation in E. coli. a-5′-end of trp mRNA containing the leader region is shown. Open bar, leader peptide; red bar-control codons. The arrows indicate the regions (1-4) that form various potential RNA hairpin structures. b- if ribosomes (shaded) complete the translation of leader peptide (i.e., there is sufficient charged tRNAtrp_{), they allow 3:4 terminator hairpin. c-if the ribosome stalls at the control codons}

because of the lack of charged tRNAtrp_{, they permit 2:3 hairpin formation, which precludes}

attenuation. (Adhya, 1999).