A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent in-frame AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397-1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397-1399 as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that astable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.     

Initiation of translation at an AUA codon for an archaebacterial protein gene expressed in E.coli.

Overexpression of the Sulfolobus solfataricus L12 ribosomal protein gene in E.coli cells yielded two products of different size. If the E.coli cells carrying the overexpression plasmid were induced in the early stage of bacterial growth, the smaller of the two products was almost exclusively produced. However, induction in a late stage of bacterial growth yielded the larger product in significant excess. The larger protein was identified as the translation product of the entire SsoL12 gene, while the smaller product was a N-terminally shortened version of the L12 protein (sh-SsoL12), starting with a N-terminal methionine at position 22 of the coded protein and continuing with the predicted protein sequence. Position 22 is an isoleucine in the complete SsoL12 protein sequence, coded by an AUA codon. A subclone (SsoL12**) of the SsoL12 gene containing overexpression plasmid, lacking the regular AUG start codon and the putative Shine Dalgarno sequence, was constructed to determine if E.coli ribosomes could initiate at this AUA codon. During overexpression the SsoL12** construct yielded exclusively the sh-SsoL12 product in significant amounts. An AUA start codon has never been found before in a natural message. However, experiments utilizing site directed mutagenesis to generate AUA start codons showed that this codon can be functional for initiation in prokaryotes and eukaryotes. The findings presented in this paper show that AUA acts as an initiation codon in a natural message expressed in a heterologous organism.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.     

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.     

Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin.

Recombinant plasmids that direct synthesis of rat preproinsulin under the direction of the SV40 early promoter have been used to probe the mechanism of initiation of translation. Insertion of an upstream AUG triplet that was out-of-frame with respect to the coding sequence for preproinsulin reduced the yield of proinsulin, in keeping with the predictions of the scanning model. The extent to which an upstream AUG codon interfered depended on sequences surrounding the AUG triplet; with two constructs ( p255 /20 and C2) the 5'-proximal AUG codon constituted an absolute barrier: there was no initiation at the downstream start site for preproinsulin. With two other constructs ( p255 /9, p255 /21), however, proinsulin was made despite the presence of an upstream, out-of-frame AUG codon in a favorable context for initiation. In those cases the reading frame set by the first AUG triplet was short, terminating before the start of the preproinsulin coding sequence. The interpretation that ribosomes initiate at the first AUG, terminate, and then reinitiate at the AUG that directly precedes the preproinsulin coding sequence was tested by introducing a point mutation that eliminated the terminator codon: the resulting mutant made no proinsulin.     

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.     

Identification of a thermostable and enantioselective amidase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an amidase expressed from the putative amidase gene (ST0478) selected from the total genome analysis from the thermoacidophilic archaeon, Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as an insoluble aggregated 6 x His-tagged fusion protein in Escherichia coli. The protein was purified with denaturing, refolding on affinity column chromatography, size exclusion filtration, and heat treatment. The enzyme exhibited high thermostability and the optimum activity for amide cleavage against benzamide was observed at around 75 degrees C and pH 7.0-8.0. It also showed enantioselectivity for (R,S)-2-phenylpropionamide and preferentially hydrolyzed the S-enantiomer. This novel enzyme is the second characterized archaeal amidase.     

Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product

It has been known that enzyme activity associated with the yeast LEU1 and LEU2 gene product (beta-isopropylmalate dehydrogenase) drops sharply when yeast is grown in the presence of leucine. RNA blot hybridizations with LEU2-specific probes establish that this is accompanied by a 5-fold repression in LEU2 mRNA levels. A similar repression was noted recently for LEU1 mRNA levels (Hsu, Y.-P., and Schimmel, P. (1984) J. Biol. Chem. 259, 3714-3719). Nuclease mapping of the 5'-end of the LEU2 mRNA shows a major start at approximately 16 nucleotides upstream of the AUG initiation codon. This initiation site in the gene is retained in an extensive LEU2 5'-noncoding region deletion which still expresses the LEU2 gene product (Erhart, E., and Hollenberg, C. P. (1983) J. Bacteriol. 156, 625-635). The primary structure of the LEU2 gene product was established from the nucleotide sequence of the gene-coding region and from fitting amino acid sequences of scattered internal peptides to the nucleotide sequence. The 364-amino acid protein has a 13-amino acid stretch which is highly homologous to the partially sequenced yeast LEU1 gene product (isopropylmalate isomerase). The homology occurs about 290 amino acids from the respective NH2 termini of the two proteins. The homology may represent residues which interact with beta-isopropylmalate, a common ligand for the enzymes.     

Increasing in archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability

UDP-GlcNAc, an activated and essential form of GlcNAc which is an important component in the polysaccharide structure of most organisms, is synthesized from GlcNAc-1-P and UTP by GlcNAc-1-P UTase. We previously reported the identification of the extremely thermostable ST0452 protein, which has dual sugar-1-P NTase activities (Glc-1-P TTase and GlcNAc-1-P UTase activities) from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Detailed analyses of the protein indicated that the activity is slightly lower than that of bacteria. For industrial applications, activity needs to be increased without decreasing thermostability. Therefore, to enhance this activity, we introduced mutations into the amino acid residues located within the predicted reaction centre by targeted mutagenesis. All 12 mutant ST0452 proteins showed no decrease in thermostability. Among them, six mutant proteins were found to have increased GlcNAc-1-P UTase activity under optimal reaction conditions with sufficient substrates or an appropriate metal ion. Our results indicate that targeted mutagenesis is a powerful technique for in vitro production of a thermostable enzyme with enhanced activity. The results of this study also indicate that the space for the metal ion is important for selecting the type of metal ion and also affects the rate of the reaction.     

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.