Our results demonstrate that when the Qβ coat gene Shine–Dalgarno sequence was occluded by stable hairpin stem structure, translational initiation from the upstream maturation cistron was activated in cis. The secondary structure at the coat gene Shine–Dalgarno site had no effect on the translation of a maturation gene present on a different RNA molecule. There was a direct correlation between the stability of a hairpin stem structure that sequestered the coat gene initiation site and the degree of maturation gene activation. Furthermore, the inhibitory effect of the coat gene initiation site on maturation gene synthesis did not require either an AUG initiator codon or initiation of coat protein synthesis.
The data are in agreement with previous observations (8
). During infection, the Qβ maturation gene is generally kept silent by extensive long-range secondary structure (27
). In fact, the thermodynamic stability of the entire 5′ domain of Qβ RNA is such that there are essentially no viable alternative competing structures predicted (27
). The single-strandedness of the coat gene initiation region renders it an extremely strong ribosome binding site relative to the much weaker maturation gene initiation site (12
). However, despite the long-range structural interaction in the 5′ domain, when Qβ replicase binds the coat gene initiation site in trans
to repress coat protein synthesis, the maturation gene becomes activated (8
). Because maturation protein mediates host cell lysis (35
), this mechanism is likely an efficient means of generating increased amounts of maturation protein when it is required late in infection.
In our experiments, when the coat gene Shine–Dalgarno site was sequestered in a stable hairpin structure to repress coat protein synthesis, both the replicase and maturation genes were activated. Presumably under these conditions, ribosomes were physically inhibited from binding to the coat gene Shine–Dalgarno site. Previously, we proposed a mechanism in which multiple translational initiation sites that have different ribosome binding strengths will compete for association with a single ribosome in cis
at any moment in time (8
). It is likely that, in the current experiments using the maturation gene plasmids, the progressive decrease in the single-strandedness at the coat gene initiation site caused a corresponding alteration in the relative ribosome binding affinities between the coat and maturation gene sites. Consequently, the probability that a ribosome would bind the less accessible upstream maturation gene site was increased. We propose the possibility that the Qβ coat and maturation gene ribosome binding sites compete in cis
for ribosome binding as a means of regulating differential protein synthesis.
Intramolecular competition for ribosome binding could be a general mechanism which would allow a very weak ribosome binding site on any polycistronic RNA to become activated whenever a stronger site within the same molecule is rendered incapable of accessing ribosomes. Indeed, we have demonstrated that the Qβ replicase gene is activated when the coat gene initiation site is either blocked or eliminated (8
), indicating that replicase expression does not need to be coupled to coat gene expression as previously thought (8
). We have further noticed that if we eliminate the coat gene ribosome binding sequence from Qβ cDNA and incorporate a strong heterologous ribosome binding site from the bacteriophage T7 gene10 (36
) 2.3-kb downstream of the maturation gene initiation site, maturation protein can be synthesized from the encoded Qβ RNA transcripts (unpublished results). Based on our observations, we suggest that there might not be anything inherent in the Qβ phage RNA coat gene initiation region that specifically leads to translational repression of the maturation gene. Instead, translational inhibition of one cistron by the presence of a strong distal ribosome binding site is likely a general mechanism that might apply to any prokaryotic polycistronic messenger RNA.
It should be mentioned that wild-type Qβ bacteriophage RNA has three ribosome binding sites. Whereas the coat gene Shine–Dalgarno site has the strongest affinity for ribosomes, the maturation gene site has the weakest. Consider that during active Qβ phage infection, coat protein needs to be made early and in large quantities. At first, translation through the coat gene region opens the replicase gene initiation site and allows translation of the replicase protein (10
). Later in infection, excess replicase protein binds and represses translation of the coat cistron, and excess coat protein binds and represses translation of the replicase cistron (38
). Elimination of both coat and replicase Shine–Dalgarno sites leaves only the maturation gene inititation site for ribosomes to access.
Such a mechanism in which a ribosome will bind at one particular translational initiation site and not another on the same mRNA would necessarily rely on a number of factors. These include the fixed distance between two ribosome binding sites on the same molecule, the dynamic equilibrium association of a 30S ribosome at a Shine–Dalgarno site, and the differential binding affinities of competing Shine–Dalgarno sites for a 30S ribosome. We will consider each of these factors below.
Fixed distance between two ribosome binding sites
Because the distance between two ribosome binding sites on the same RNA is a constant, these sites can be considered to be at high concentration relative to one another, and independent of the cellular message RNA concentration. However, when two sites lie on separate molecules, their concentration is a function of the cellular mRNA concentration. Consequently, the relative concentration of any two ribosome binding sites with respect to a single ribosome is dependent upon whether or not the two sites are present on the same molecule. Consider a volume of a cell in which free mRNAs are equally distributed, and mRNA concentration is a function of the number of RNA molecules present. Under these conditions, unbound ribosomes would be distributed proportionally among free mRNAs. If each RNA molecule contained only one Shine–Dalgarno sequence, and these had equal ribosome binding affinities, then ribosome association and translation would be proportional to the concentration of messenger RNAs.
Alternatively, when two different Shine–Dalgarno sites are present in cis on the same polycistronic message, the situation is very different. The concentration of the intramolecular sites relative to one another is now independent of cell volume. Instead, it is a constant that is determined by a fixed distance between the two sites. This distance would be determined both by the number of nucleotides between the two sites, and by RNA structure that can bring the two sites into closer proximity. Hence, the concentration of two ribosome binding sites relative to one another can be extremely high compared with that of available ribosomes. As such, the local concentration of unbound 30S ribosomes would always be limiting with respect to these two sites, regardless of the ribosome or mRNA concentration in the cell. The immediate reaction then becomes that of two ribosome binding sites ‘competing’ for association with only one ribosome. Note that this competition model considers available ribosomes only, and not those already involved in translational elongation throughout the cell.
Dynamic equilibrium of a 30S ribosome complex
It is generally accepted that because ribosomes in a cell are usually present in excess of message RNA molecules, all accessible ribosome binding sites can be saturated. However, binary complex association is a dynamic reversible equilibrium process (31
), and so the 30S ribosome is never permanently bound at any one Shine–Dalgarno site. Following the association at a Shine–Dalgarno locus, the 30S ribosomal subunit will proceed in one of two ways: either it will translocate to the initiator codon and undergo protein synthesis, thereby eliminating itself from the pool of unbound ribosomes; or it will dissociate from the RNA. As such, Shine–Dalgarno sites would never be completely saturated at any given moment, but would be continually accessible for 30S ribosome binding.
Differential affinities of ribosome binding sites
Several factors contribute to the ribosome binding affinity of a Shine–Dalgarno region. Among these are: the degree of complementarity between a Shine–Dalgarno region and 16S ribosomal RNA (2
); the secondary structure that comprises the Shine–Dalgarno region (30
); the presence of either a nearby ribosomal protein S1 binding site or an enhancer site on the mRNA (40–42
); the presence of putative standby sites for 30S ribosomes close to a Shine–Dalgarno sequence (13
); and the putative interaction of a trans
-acting protein or RNA that can bind a message RNA to block ribosome access (8
). Hence, when two Shine–Dalgarno sites are present on the same polycistronic message, the probability that a single ribosome will associate with one or the other site is determined by their relative affinities for a 30S ribosome. The greater the difference in binding affinities, the more dramatic would be the competition between the two sites.
General implications of intramolecular competition between two ribosome sites
Our findings suggest that all polycistronic translational systems might be affected to some degree by competition in cis
between multiple ribosome entry sites. Since competition within a single RNA molecule is putatively independent of both mRNA and ribosome concentration, all mRNAs carry the potential for this type of translational regulation. The more extreme the differences are in ribosome binding affinities among multiple sites on an RNA message, the more profound would be the regulatory effect. Competition in cis
would not only affect the translational balance between multiple cistrons within a polycistronic mRNA, but also the possibility exists that pseudo-ribosome entry sites affect the efficiency of translational initiations at one or more genes on any given messenger RNA. Although association between a 30S ribosome and a messenger RNA depends upon a number of factors (see above), the 30S:mRNA association does not require an AUG initiator codon. As such, a translational initiation site might be rendered inactive simply because it is inhibited by a second, more competitive pseudo-ribosome binding site present within the same RNA molecule. Indeed, even ribosome binding sites that appear to be silent due to long-range secondary interactions are capable of accessing ribosomes in the absence of stronger competing sites. For example, the Qβ maturation gene was once believed to be completely inactive because of long-range secondary structure, but appears to be expressed to a maximum in the absence of the stronger downstream coat gene initiation site (8
). We have previously proposed the possibility that for a large folded RNA domain, the kinetics of folding and re-folding can be very slow, thus allowing occasional exposure of a translational initiation site (8
The effect of a putative competing ribosome entry site on distal gene translation might be modulated by several factors, such as: coupled translation with a second gene; interaction with either a trans-
acting protein factor, or with antisense RNA (9
); processing of an RNA into two or more separate mRNA molecules; or the formation of alternate RNA conformations. Consequently, the proposed mechanism of competition in cis
would enable a single polycistronic mRNA to exist as one of two or more different functional messenger RNAs, each capable of translating a different proportion of the same encoded proteins. Such a process would provide a sophisticated means of translational auto-regulation not necessarily confined to the RNA coliphages. Since RNA phage genomes are highly adapted to utilizing the host translational apparatus, it is possible that other bacterial messenger RNA systems usefully employ a similar regulatory mechanism. It has been shown that prokaryotic polycistronic mRNAs can generate different proteins in quantities that vary over three orders of magnitude (3
). Consequently, it is crucial to understand how alternative regulatory mechanisms govern the differential synthesis of multiple protein products from these RNA messages.
Many intriguing systems exist in which potential ribosome entry site competition might influence prokaryotic translation in cis
. For example, there are intragenic ribosome entry sites that have been shown to affect gene expression (45
). Mechanisms also exist that are responsible for masking independent initiation of translation (47–49
). Translational competition has been shown to occur between one or more cistrons that are fused to a reporter gene within an RNA message (50
). Translation of some cellular genes might be selectively enhanced by trans-
acting repressor proteins, e.g. the T4 RegA
). In the E. coli
rpmI-rplT operon encoding ribosomal proteins L35 and L20, a kinetic model is suggested in which the L20 repressor protein competes with 30S ribosomes for binding at the operator region to regulate translation (52
). In addition, studies in eukaryotic systems suggest that translational regulation of human fibroblast growth factor might be affected by competition between a cap-dependent translational mechanism and an internal ribosome entry site-dependent mechanism (53
In conclusion, we suggest that the following points should be considered with respect to translational control mechanisms: (i) all ribosome entry sites on a single messenger RNA can compete in cis for a single 30S ribosome; (ii) these ribosome entry sites are never saturated with ribosomes at any instant in time; (iii) competition in cis can occur when the cellular messenger RNA concentration is extremely low relative to the local concentration of two ribosome binding sites on the same messenger RNA; and (iv) reference genes that are inserted into a messenger RNA molecule might significantly influence translational initiations that occur at a distal experimental gene. Each of these points should be carefully considered when conducting experiments with cis-acting reporter genes and truncated message RNA molecules. It might be necessary to carry out such studies using intact mRNA molecules in the presence of any trans-acting RNA binding proteins, or antisense RNA transcripts that could influence gene expression.