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Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.