Genes VII

Translational control is a notable feature of operons coding for components of the protein synthetic apparatus. The operon provides an arrangement for coordinate regulation of a group of structural genes. But, superimposed on it, further controls, such as those at the level of translation, may create differences in the extent to which individual genes are expressed.

A similar type of mechanism is used to achieve translational control in several systems. Repressor function is provided by a protein that binds to a target region on mRNA to prevent ribosomes from recognizing the initiation region. Formally this is equivalent to a repressor protein binding to DNA to prevent RNA polymerase from utilizing a promoter. Figure 10.31 illustrates the most common form of this interaction, in which the regulator protein binds directly to a sequence that includes the AUG initiation codon, thereby preventing the ribosome from binding.

Some examples of translational repressors and their targets are summarized in Figure 10.32. A classic example is the coat protein of the RNA phage R17; it binds to a hairpin that encompasses the ribosome binding site in the phage mRNA. Similarly the T4 RegA protein binds to a consensus sequence that includes the AUG initiation codon in several T4 early mRNAs; and T4 DNA polymerase binds to a sequence in its own mRNA that includes the Shine-Dalgarno element needed for ribosome binding.

Another form of translational control occurs when translation of one cistron requires changes in secondary structure that depend on translation of a preceding cistron. This happens during translation of the RNA phages, whose cistrons always are expressed in a set order. Figure 10.33 shows that the phage RNA takes up a secondary structure in which only one initiation sequence is accessible; the second cannot be recognized by ribosomes because it is base paired with other regions of the RNA. However, translation of the first cistron disrupts the secondary structure, allowing ribosomes to bind to the initiation site of the next cistron. In this mRNA, secondary structure controls translatability.

About 70 or so proteins constitute the apparatus for bacterial gene expression. The ribosomal proteins are the major component, together with the ancillary proteins involved in protein synthesis. The subunits of RNA polymerase and its accessory factors make up the remainder. The genes coding for ribosomal proteins, protein-synthesis factors, and RNA polymerase subunits all are intermingled and organized into a small number of operons. Most of these proteins are represented only by single genes in E. coli.

Coordinate controls ensure that these proteins are synthesized in amounts appropriate for the growth conditions: when bacteria grow more rapidly, they devote a greater proportion of their efforts to the production of the apparatus for gene expression. An array of mechanisms is used to control the expression of the genes coding for this apparatus and to ensure that the proteins are synthesized at comparable levels that are related to the levels of the rRNAs.

The organization of six operons is summarized in Figure 10.34. About half of the genes for ribosomal proteins (r-proteins) map in four operons that lie close together (named str, spc, S10, and α simply for the first one of the functions to have been identified in each case). The rif and L11 operons lie together at another location.

Each operon contains a mélange of functions. The str operon has genes for small subunit ribosomal proteins as well as for EF-Tu and EF-G. The spc and S10 operons have genes interspersed for both small and large ribosomal subunit proteins. The α operon has genes for proteins of both ribosomal subunits as well as for the α subunit of RNA polymerase. The rif locus has genes for large subunit ribosomal proteins and for the β and β′ subunits of RNA polymerase.

All except one of the ribosomal proteins are needed in equimolar amounts, which must be coordinated with the level of rRNA. The dispersion of genes whose products must be equimolar, and their intermingling with genes whose products are needed in different amounts, pose some interesting problems for coordinate regulation.

A feature common to all of the operons described in Figure 10.34 is regulation of some of the genes by one of the products. In each case, the gene coding for the regulatory product is itself one of the targets for regulation. Autogenous regulation occurs whenever a protein (or RNA) regulates its own production. In the case of the r-protein operons, the regulatory protein inhibits expression of a contiguous set of genes within the operon, so this is an example of negative autogenous regulation.

In each case, accumulation of the protein inhibits further synthesis of itself and of some other gene products. The effect often is exercised at the level of translation of the polycistronic mRNA. Each of the regulators is a ribosomal protein that binds directly to rRNA. Its effect on translation is a result of its ability also to bind to its own mRNA. The sites on mRNA at which these proteins bind either overlap the sequence where translation is initiated or lie nearby and probably influence the accessibility of the initiation site by inducing conformational changes. For example, in the S10 operon, protein L4 acts at the very start of the mRNA to inhibit translation of S10 and the subsequent genes. The inhibition may result from a simple block to ribosome access, as illustrated previously in Figure 10.32, or it may prevent a subsequent stage of translation. In two cases (including S4 in the α operon), the regulatory protein stabilizes a particular secondary structure in the mRNA that prevents the initiation reaction from continuing after the 30S subunit has bound (Baughman and Nomura, 1983).

The use of r-proteins that bind rRNA to establish autogenous regulation immediately suggests that this provides a mechanism to link r-protein synthesis to rRNA synthesis. A generalized model is depicted in Figure 10.35. Suppose that the binding sites for the autogenous regulator r-proteins on rRNA are much stronger than those on the mRNAs. Then so long as any free rRNA is available, the newly synthesized r-proteins will associate with it to start ribosome assembly. There will be no free r-protein available to bind to the mRNA, so its translation will continue. But as soon as the synthesis of rRNA slows or stops, free r-proteins begin to accumulate. Then they are available to bind their mRNAs, repressing further translation. This circuit ensures that each r-protein operon responds in the same way to the level of rRNA: as soon as there is an excess of r-protein relative to rRNA, synthesis of the protein is repressed (for review see Nomura et al., 1984).

Autogenous regulation has been placed on a quantitative basis for gene 32 of phage T4. The protein (p32) plays a central role in genetic recombination, DNA repair, and replication, in which its function is exercised by virtue of its ability to bind to single-stranded DNA. Nonsense mutations cause the inactive protein to be overproduced. So when the function of the protein is prevented, more of it is made. This effect occurs at the level of translation; the gene 32 mRNA is stable, and remains so irrespective of the behavior of the protein product.

Figure 10.36 presents a model for the gene 32 control circuit. When single-stranded DNA is present in the phage-infected cell, it sequesters p32. However, in the absence of single-stranded DNA, or at least in conditions in which there is a surplus of p32, the protein prevents translation of its own mRNA. The effect is mediated directly by p32 binding to mRNA to prevent initiation of translation. Probably this occurs at an A PT-rich region that surrounds the ribosome binding site.

Two features of the binding of p32 to the site on mRNA are required to make the control loop work effectively:

• The affinity of p32 for the site on gene 32 mRNA must be significantly lower than its affinity for single-stranded DNA. The equilibrium constant for binding RNA is in fact almost two orders of magnitude below that for single-stranded DNA.
• But the affinity of p32 for the mRNA must be significantly greater than the affinity for other RNA sequences. It is influenced by base composition and by secondary structure; an important aspect of the binding to gene 32 mRNA is that the regulatory region has an extended sequence lacking secondary structure.

Using the known equilibrium constants, we can plot the binding of p32 to its target sites as a function of protein concentration. Figure 10.37 shows that at concentrations below 10 ^V6 M, p32 binds to single-stranded DNA. At concentrations >10 ^V6 M, it binds to gene 32 mRNA. At yet greater concentrations, it binds to other mRNA sequences, with a range of affinities.

These results imply that the level of p32 should be autoregulated to be <10 ^V6 M, which corresponds to ~2000 molecules per bacterium. This fits well with the measured level, 1000 V2000 molecules/cell.

A feature of autogenous control is that each regulatory interaction is unique: a protein acts only on the mRNA responsible for its own synthesis. Phage T4 provides an example of a more general translational regulator, coded by the gene regA, which represses the expression of several genes that are transcribed during early infection. RegA protein prevents the translation of mRNAs for these genes by competing with 30S subunits for the initiation sites on the mRNA. Its action is a direct counterpart to the function of a repressor protein that binds multiple operators.

Autogenous regulation is a common type of control among proteins that are incorporated into macromolecular assemblies. The assembled particle itself may be unsuitable as a regulator, because it is too large, too numerous, or too restricted in its location. But the need for synthesis of its components may be reflected in the pool of free precursor subunits. If the assembly pathway is blocked for any reason, free subunits accumulate and shut off the unnecessary synthesis of further components (for review see Gold, 1988).

Eukaryotic cells have a common system in which autogenous regulation of this type occurs. Tubulin is the monomer from which microtubules, a major filamentous system of all eukaryotic cells, are synthesized. The production of tubulin mRNA is controlled by the free tubulin pool. When this pool reaches a certain concentration, the production of further tubulin mRNA is prevented. Again, the principle is the same: tubulin sequestered into its macromolecular assembly plays no part in regulation, but the level of the free precursor pool determines whether further monomers are added to it.

The target site for regulation is a short sequence at the start of the coding region. We do not know yet what role this sequence plays, but two models are illustrated in Figure 10.38. Tubulin may bind directly to the mRNA; or it may bind to the nascent polypeptide representing this region. Whichever model applies, excess tubulin causes tubulin mRNA that is located on polysomes to be degraded, so the consequence of the reaction is to make the tubulin mRNA unstable.

Autogenous control is an intrinsically self-limiting system, by contrast with the extrinsic control that we discussed previously. A repressor protein’s ability to bind an operator may be controlled by the level of an extraneous small molecule, which activates or inhibits its activity. But in the case of autogenous regulation, the critical parameter is the concentration of the protein itself.