Polycistronic messenger

Polycistronic messenger RNA. In prokaryotes, an RNA that contains two or more cistrons note that only in prokaryotic mRNAs can more than one cis-tron be utilized by the translation system to generate individual proteins. 

S Johnston, JH Lee, DS Ray. High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA. Gene 34 137-145, 1985. 

Viral RNA, isolated from a number of plant, animal, and bacterial viruses, may be considered as a polycistronic messenger RNA. It has been shown to have molecular weights of one or two million. Generally speaking, there is one molecule of RNA per infective virus particle. The RNA of an RNA virus can be separated from its protein component and is also infective, bringing about the formation of complete virus. 

Fig. 1. Schematic diagram of transcription and translation of the lac operon. The operon contains the controlling elements p and o and the structural genes, z, y, and a. Near the p region is the i gene which produces the lac repressor protein. When induced, the lac region of the DNA is transcribed into a polycistronic lac messenger RNA, which is subsequently translated into three proteins—galactosidase, M protein, and transacetylase. The polymeric nature of the protein is indicated.

As shall be seen in Section IV, induction studies have shown that the three enz3mies of the operon are induced coordinately. Such a situation could be explained either by the production of a polycistronic messenger or alternatively by coordinate induction of three separate messenger RNA species. Kinetic arguments (discussed below) have ruled out the latter possibility. Kiho and Rich [45] have presented direct evidence in favor of a polycistronic lac messenger. They measured polysomes in... 

A polycistronic messenger RNA molecule of discrete size suggests the existence of a well-defined termination signal as well as an initiation signal. Little is known [33] concerning the nature of these hypothesized signals, although recently Roberts [49] has found a protein factor, p, which appears to participate in RNA chain termination. 

A rough estimate of the size of the histidine operon is obtained from the sedimentation velocity of the polycistronic messenger. In Martin s experiment [48], a sedimentation coefficient of 34 S was found. A more recent experiment by Venetianer et a , confirmed this figure [50]. The value of 34 S corresponds to a length of about 10,000 nucleotides in the mRNA. 

The histidine operon consists of an operator region followed by the genes coding for the enzymes of histidine biosynthesis. The genes are transcribed into a single polycistronic messenger RNA, so that enzyme activities are controlled in a coordinate fashion. Transcription of the DNA code into mRNA, and probably also translation of the mRNA... 

Although fundamentally similar in bacteria and eukaryotic cells, transcription and translation must differ in details because of the difference in structural organization and of the surrounding environment. The environment of bacterial cells may change suddenly and considerably, but that of mammalian cells is stable because of homeostatic control. Therefore, metabolic patterns are much more stable in mammalian than in bacterial cells, and ordinarily there is no need for the sudden appearance of enzyme clusters like those that appear in bacteria challenged by inducers. Thus, it may not be necessary to produce polycistronic messenger in mammalian cells. Moreover, the mammalian cell may find it advantageous to maintain stable templates. 

Hayashi and Hayashi have prepared a polycistronic messenger of the X 174 phage—a DNA phage containing approximately 10 cistrons, the functions of 9 of which have been identified [190]. Several laboratories have prepared messenger RNA from viral, bacterial, and even mammalian sources. 

This polarity suggests that the chances of transcribing the DNA sequence of the polycistronic operon into a complementary sequence of messenger RNA decrease as the DNA sequence is more distal to the operator gene. 

A molecular explanation for polarity must exist. The existence of modulating triplets has been invoked to explain polarity. The polycistronic operon dictates the biosynthesis of a single ribbon of messenger RNA. The ribosome attaches to the messenger RNA by means of the anticodons of the tRNA. Messenger and... 

The anticodon may either correspond to a triplet sequence normally involved in dictating the position of a given amino acid, or it is a modulating triplet, which facilitates the disconnection of the ribosome from the messenger. Thus, the chances for a ribosome to fall off its mRNA increase as the distance between the reading point and the operator increases. The position of the cistron within the polycistronic operon and the existence of modulating triplets could well explain the modulation of protein synthesis, or rate at which different proteins are synthesized. When the polarity is modified as a result of a mutation, it is assumed that a coding triplet is replaced by a modulation triplet. 

The template properties of virus RNA are manifested, as in the case of messenger RNA of the host cell, during its interaction with ribosomes. Just as in the case of normal intracellular synthesis, polysome structures of the type illustrated in Fig. 8 are formed under these circumstances. The formation of active polysomes during interaction between virus RNA and cell ribosomes have been studied in particular detail in the case of poliovirus infections of HeLa cell cultures (Penman et al., 1963 Scharff et al., 1963 Rich et al., 1963). In this condition virus-specific polysomes are formed which are much bigger than the polysomes of uninfected HeLa cells, as a result of the polycistron character of virus RNA. Some of them contained as many as 60 ribosomes. 

Fig. 11. Scheme of two possible methods of reading a polycistron messenger RNA. The messenger RNA in this case codes eight proteins (A-H) and the small circles represent ribosomes, a) Ribosomes are attached to each cistron and initiate independent synthesis of several proteins b) there is only one point of initial attachment for the whole RNA molecule and proteins are synthesized sequentially (Rich et al., 1963). 

It is usually assumed that the histidine operon is transcribed into one molecule of messenger RNA, which is then transcribed by the polyribosome system from one end to the other. The histidine operon and corresponding mRNA consist of about 13,000 nucleotides. The possibility that such a polycistronic histidine messenger RNA exists was demonstrated by the work of Martin (1963). The RNA fraction, labeling of which took place differently in histidine-constitutive and histidine-deficient strains, had a sedimentation... 

This can be explained either by the formation of individual forms of mRNA for each cistron, or by the ability of the integral operon (polycistron) RNA to translate the information with some degree of selectivity, i.e., by the phenomenon of regulation at the level of interaction between messenger RNA molecules and ribosomes. 

Fig. 28. Four possible models of protein synthesis under the control of polycistron op-erons. The histidine operon is shown at the top of the scheme. Capital letters denote genes (cistrons) controlling histidine biosynthesis. DNA is represented as a double helix messenger RNA as straight lines of different lei ths ribosomes as small black circles and proteins as larger black circles. The time scale is indicated in the bottom left comer of the scheme (Goldberger and Berberich, 1966).

Polycistronic forms of messenger RNA, carrying information for several proteins, as we have seen are typical of viruses and are found in bacteria during the study of synthesis of proteins coded in one operon. However, in this case also we are forced to ask whether this RNA functions in the stage of protein synthesis on polysomes as a single linear system or as a sum of independently working cistrons, producing different numbers of protein molecules. 

Inthepolycistronicoperons controlling the multistage synthesis of a metabolite such as histidine, at every stage different numbers of molecules of enzymes must be formed because the specific activity of the enzymes differs and depends both on protein structure and on the character of the reaction it catalyzes. From this aspect, the linear polarity of transcription-of information from polycistronic messenger RNA molecules of a polysome system raises the question of the mechanism responsible in this case for differences in the velocity of protein synthesis by different cis-trons running their course in the polysomes. Ames and Hartmann (1963) proposed a very interesting explanation of this phenomenon and undertook its preliminary experimental verification. 

According to their theory, as the polycistronic messenger RNA moves in relation to the polysome system, the velocity of protein synthesis in its various parts is slowed. They postulated that the sequence of the genes in the histidine operon (which does not correspond to the biochemical sequence of reactions) is connected with the number of molecules of each enzyme synthesized. By analyzing the frequency of mutations of polarity, they concluded that many triplets (of the 64 possible) can retard the transcription and translation of information. The essence of the matter is that if any nucleotide triplet (codon) XYZ requires an anticodon in the molecules of acceptor sRNA for itstranslationinto a protein "text," a lowered content of this fraction of sRNA with the corresponding anticodon may act as modulator of the velocity of translation, which is reduced at this locus in connection with a decrease in the number of codon-anticodon interactions. 

FIGURE 7.7 Comparative structures of messenger RNA. (a) Polycistronic prokaryotic RNA transcript, (b) Monocistronic eukaryotic mRNA... 

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