mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli

Codon usage and thermodynamic optimization of the 5'-end of mRNA have been applied to improve the efficiency of human protein production in Escherichia coli. However, high level expression of human protein in E. coli is still a challenge that virtually depends upon each individual target genes. Using human interleukin 10 (huIL-10) and interferon alpha (huIFN-alpha) coding sequences, we systematically analyzed the influence of several major factors on expression of human protein in E. coli. The results from huIL-10 and reinforced by huIFN-alpha showed that exposing AUG initiator codon from base-paired structure within mRNA itself significantly improved the translation of target protein, which resulted in a 10-fold higher protein expression than the wild-type genes. It was also noted that translation process was not affected by the retained short-range stem-loop structure at Shine-Dalgarno (SD) sequences. On the other hand, codon-optimized constructs of huIL-10 showed unimproved levels of protein expression, on the contrary, led to a remarkable RNA degradation. Our study demonstrates that exposure of AUG initiator codon from long-range intra-strand secondary structure at 5'-end of mRNA may be used as a general strategy for human protein production in E. coli.     

Structure of the Escherichia coli S10 ribosomal protein operon.

The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.     

Feedback regulation of the rplJL-rpoBC ribosomal protein operon of Escherichia coli requires a region of mRNA secondary structure

The rplJ-rpoBC (L10) operon of Escherichia coli is regulated in part through translational repression (feedback regulation) by ribosomal protein L10 or a complex of ribosomal proteins L10 and L7/L12 (L10-L7/L12). We have constructed mutants in the untranslated leader region of a rplJ-lacZ fusion by oligonucleotide-directed mutagenesis. The mutations include several deletions and a number of single base changes, all of which fail to exhibit normal feedback regulation. Chemical probing of part of the rplJ mRNA leader in the mutagenized region confirms that all of the mutations lie in a stem structure located 140 nucleotides upstream from the translation start-site. The structure includes a 12 base-pair stem, a four base stem-loop, and a six base bulge-loop. Point mutations that abolish feedback regulation are presumed to disrupt this stem structure. Pseudorevertants of selected point mutations were constructed by combining pairs of single base mutations. In these cases, both the secondary structure of the RNA and feedback regulation were restored. The results allow us to define a region of secondary structure in the rplJ mRNA leader that is necessary for feedback regulation.     

Deletion of a ribosomal ribonucleic acid operon in Escherichia coli.

One (rrnE) of the seven operons which codes for ribosomal ribonucleic acid in Escherichia coli was deleted. No significant change in phenotype was observed even under maximum laboratory growth conditions.     

Polarity of amber mutations in ribosomal protein genes of Escherichia coli.

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.     

Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli.

The L11 operon in Escherichia coli consists of the genes coding for ribosomal proteins L11 and L1. It is known that translation of L1 does not take place unless the preceding L11 cistron is translated, that is, the two cistrons are translationally coupled, and this is the basis of coregulation of the translation of the two cistrons by a single repressor, L1. Several mutational analyses were carried out to define the region responsible for coupling L1 translation with L11 translation. First, by introducing several amber mutations into the L11 gene by a site-directed mutagenesis technique, it was shown that translation by ribosomes down to a position 21 nucleotides upstream, but not to a position 45 nucleotides upstream, from the end of the L11 cistron allowed the initiation of L11 translation. Second, deletion analysis indicated that a region located 23 to 20 nucleotides from the end of the L11 gene was involved in preventing independent initiation from L1 translation. Third, five different mutations obtained by screening for activation of the masked L1 initiation site were found to be clustered in a small region immediately upstream from the Shine-Dalgarno sequence of L1, and all of them were G-to-A transitions. These results, together with some additional experiments with oligonucleotide-directed mutagenesis, defined the region involved in the coupling and suggest that some special feature of this region, probably different from simple masking of the initiation site by base pairing, is responsible for translational coupling. The present results also suggest that there might be specific differences in the primary nucleotide sequence that distinguish independent translational initiation sites from translationally coupled (i.e., masked) initiation sites.     

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression

Summary A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.