Translational Control In Prokaryotes

16.1. General Considerations

Protein production in the cell can be controlled principally at three levels: (1) by production of mRNA (transcriptional level), (2) through availability of mRNA for translation and modulation of mRNA translation rate (translational level), and (3) by mRNA elimination (degradation). Although both transcription and mRNA degradation may also depend on ribosomes, only the translational level of protein synthesis regulation will be considered here as the theme directly relevant to the object of the book.

The main ways of translational regulation of protein synthesis are accomplished through the control of translation initiation. Under certain circumstances, translation of an individual mRNA or a cistron within a polycistronic mRNA may be or may not be started; this case can be classified as an all-or-none control of initiation. When initiation is principally permitted, the rate of initiation is different for various mRNAs, that is mRNAs display differential "strength" in their entering into initiation process. Furthermore, the rate of initiation, totally or for individual mRNAs, can be modulated in a wide range by internal or external signals thus determining the modulation of protein production of the cell. Both Prokaryotes and Eukaryotes possess well developed systems of the translational regulation through the control of initiation. At the same time, the two groups of organisms exhibit so principal differences in the mechanisms of their translation initiation and its control that it is worthwhile to consider them separately.

It is generally accepted that in Prokaryotes protein synthesis is controlled mainly at the level of transcription. Indeed, metabolic instability of mRNA in prokaryotic cells, involving its rapid synthesis and rapid degradation, provides for a fast change of templates depending on environmental conditions and cell requirements. At the same time, however, the existence of polycistronic templates in Prokaryotes often demands differential control of the individual cistron activities in order to provide for quantitatively different or temporary uncoupled production of proteins encoded by a given polynucleotide. Moreover, in a number of cases the accumulation of excessive amounts of the product of translation may be used to shut down the translation of corresponding mRNA (autoregulation); in this way, a very fine tuning between the level of protein production and the extent of cell requirement in this protein can be achieved. Thus, the translational level of regulation of protein synthesis in Prokaryotes may be of great importance in many special cases (for reviews, see, e.g., Stormo, 1987; Gold, 1988; McCarthy & Gualerzi, 1990; Voorma, 1996), though the general pattern of protein production seems to be determined mostly by the activities of genes, i.e. at the transcriptional level.

16.2. Discrimination of mRNAs

The discrimination of mRNAs by initiating ribosomal particles is typical of Prokaryotes. The prokaryotic 30S ribosomal particle recognises the structural element called ribosome-binding site (RBS) containing Shine-Dalgarno sequence and initiation codon (Section 15.2.2). The primary structure and the availability of this region for the interaction with the initiating ribosomal particle are of primary importance for prokaryotic initiation "strength" (for reviews, see Steitz, 1980; Stormo, 1986; Gold, 1988; Gualerzi et al., 1990; de Smit & van Duin, 1990).

The availability of the RBS depends, first of all, on mRNA secondary and tertiary structure. The intramolecular mRNA folds involving Shine-Dalgarno sequence and/or initiation codon, if they are stable enough, can completely block the access of ribosomal particles to the initiation site on mRNA (until some competing interactions melt the fold). In a more common case, the stability is not so high, and the availability of RBS for ribosomal particles will be determined by the competition between the intramolecular secondary/tertiary structure formation and the ribosome-mRNA interaction. In any case, the existence of a secondary or tertiary structure in the RBS region seems to always reduce the initiation rate. The more stable the fold, the more reduction is expected. In the region upstream the initiation codon and the Shine-Dalgarno sequence, the absence or low stability of secondary/tertiary structure may also contribute to higher initiation rate. At the same time, however, some structural elements around or inside the RBS can facilitate the ribosome binding to mRNA - e.g., by better exposing the Shine-Dalgarno sequence and/or the initiation codon, or by better adjusting the distance between them.

The affinity o f the 30S ribosomal particle for the available RBS depends on the degree of complementarity between the Shine-Dalgarno sequence and the 16S RNA 3'-terminal sequence. The distance between the Shine-Dalgarno sequence and initiation codon, the nature of initiation codon (AUG or non-AUG), and other structural environments may also contribute to the affinity. Generally, a higher affinity provides a higher initiation rate. Concerning the Shine-Dalgarno sequence contribution, the most frequent situation among bacterial mRNAs is four to six base pairs (A:U and G:C) between the mRNA sequence and the 16S RNA terminus. Mutations in the Shine-Dalgarno sequences of mRNAs that decrease the complementarity with the 16S RNA do reduce the initiation rate. Longer Shine-Dalgarno sequences usually provide better initiation rate. When the spacing between the Shine-Dalgarno sequence and the initiation codon is more than 12 nucleotides or less than 5 nucleotides, the initiation rate is also decreased. AUG can be qualified as the strongest initiation codon among other initiation codons (GUG, UUG, etc.). Weak initiation was reported for rare initiation codons, such as AUA and AUU. Some special sequences (initiation "enhancers") upstream of the Shine-Dalgarno sequence may facilitate the initiation rate also kinetically, probably by complementarily interacting with exposed regions of the ribosomal RNA of the 30S subunit; they are believed to fish out the initiating particles from the surroundings and attract them to the proper mRNA sites (McCarthy & Brimacombe, 1994). Thus, different mRNAs have different capacities to bind initiating ribosomal subunits. Hence, when they compete, the strongest win.

Indeed, in bacterial cells some mRNAs are much higher expressible than others, due to their higher initiation rates. These are primarily the mRNAs that encode for abundant proteins of the bacterial cell, such as two major outer membrane proteins, OmpA and lipoprotein, in Escherichia coli, the multiple c-subunit of proton-translocating ATPase of plasma membrane, ribosomal proteins, elongation factors EF-Tu and EF-G. As expected, bacteriophage RNAs encoding for coat proteins are especially highly expressible. A special emphasis should be made on some highly expressible (very strong) bacteriophage mRNAs, the mRNA transcribed from bacteriophage T7 gene 10 being one of the best studied among them.

The synthesis of eight types of protein subunits of proton-translocating ATPase (a3p3Y181e1a1b2c10_15) is a good example how the differential efficiencies of translational initiation at different mRNAs correspond to the required subunit stoichiometry in the final enzymatic complex (McCarthy, 1988, 1990). Five types of the subunits (a, b, g, 8 and e) form the soluble part (Fj) of the enzyme, whereas the membrane-bound part (F0) comprises three types of subunits (a, b and c). All the mRNAs for the ATPase subunits are the sections of the same polycistronic transcript (which starts with the message I for unknown protein), and therefore the amount of messages is equal for each subunit:

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