Thomas C. Gluick

Translational Repression of the Escherichia coli α Operon mRNA IMPORTANCE OF AN mRNA CONFORMATIONAL SWITCH AND A TERNARY ENTRAPMENT COMPLEX

Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in theEscherichia coli α operon by binding to a nested pseudoknot structure that spans the ribosome binding site. A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are “active” or “inactive” in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA (“entrapment”), in contrast to direct competition between repressor and ribosome binding (“displacement”). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities. S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits. Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the α operon by an entrapment mechanism.

Pseudoknots, RNA Folding, and Translational Regulation

The primary function of messenger RNAs is to encode genetic information, but mRNAs also encode signals that modulate translational efficiency and regulate gene expression. These signals include sequences and structures that define processing sites in higher organisms, enhance the probability of frameshift events or stop codon readthrough, and serve as targets for translational repressors. The concern of this review is the phenomenon of specific protein-mRNA interactions that regulate translational initiation. The first example of a protein translational repressor was found in RNA phages, where the phage coat protein binds to the translational initiation site for the replicase gene and shuts down replicase synthesis late in phage infection (Nathans et al. 1969; Spahr et al. 1969). Subsequently, a number of examples of translational regulation were discovered in T4 phage, including autoregulation of gene 32 and gene 43 (DNA polymerase), and regulation of a number of genes by the regA gene product (Gold 1988). About the same time, it was realized that Escherichia coli makes extensive use of translational feedback regulation to match ribosomal protein synthesis to the rate of ribosomal RNA synthesis (Nomura et al. 1984). Other E. coli proteins are now known to regulate their own synthesis at the translational level also. Repression at the transcriptional level is frequently a simple matter of competition between RNA polymerase and a repressor protein for binding to overlapping sites, as recently demonstrated for the lac operon (Schlax et al. 1995). The DNA tends to be a passive participant in this process, providing only...

Thermal Methods for the Analysis of RNA Folding Pathways

Once a model of the secondary structure of an RNA has been deduced, thermal melting analysis can be used to determine whether the model accounts for all intramolecular interactions of the RNA, or whether noncanonical and tertiary interactions make the structure more stable than predicted, or link parts of the structure in unexpected ways. It is also useful to determine the pH, salt, and temperature ranges under which the RNA adopts a stably folded structure, or to analyze unfolding pathways. This unit discusses sample preparation, instrumentation, and theoretical background. It also provide a sample analysis of tRNA unfolding.

Tertiary structure of ribosomal RNA

Abstract The secondary structures of ribosomal RNAs have been deduced by comparative sequence analysis. The same approach has also suggested some higher-order tertiary interactions. Some of these are now being tested by direct experiment, and it is clear that tertiary interactions play an important functional role in stabilizing protein-binding sites and folding the ribosomal RNAs into their functional forms. At the same time, diverse experimental data on the relative positioning of RNA segments and proteins within the ribosome are being incorporated into low-resolution models of the ribosome, which reveal a distinctive domain organization of the RNA.