Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin
which means one. Figure 2.34 shows a typical polycistronic mRNA in which the coding sequence for one polypeptide is followed by the coding sequence for another. The space between two coding regions can be very short, and the coding sequences may even overlap. For example, the coding region for one polypeptide may end with the termination codon UAA, but the last A may be the first nucleotide of the initiator codon AUG for the next coding region. Even if the two coding regions overlap, the two polypeptides on an mRNA can be translated independently by different ribosomes.
Polycistronic mRNAs do not exist in eukaryotes, in which, as described above, TIRs are much less well defined and translation usually initiates at the AUG codon closest to the 5′ end of the mRNA. In eukaryotes, the synthesis of more than one polypeptide from the same mRNA usually results from differential splicing of the mRNA or from high-level frameshifting during the translation of one of the coding sequences (see “Reading Frames” above); there are also specialized events in which an RNA element called an internal ribosome entry sequence directs binding of a ribosome to a site within the RNA. Polycistronic RNA leads to phenomena unique to bacteria, i.e., translational coupling and polarity, which are described below.
Figure 2.34 Structure of a polycistronic mRNA. (A) The coding sequence for each polypeptide is between the initiation codon and the stop codon. The region 5′ of the first initiation codon is called the leader sequence, and the untranslated region between a stop codon for one gene and the next initiation codon is known as the intercistronic spacer. (B) The association of the 30S and 50S ribosomes at a translational initiation region (TIR) and their dissociation at a stop codon. New 30S and 50S subunits associate at a downstream TIR.
TRANSLATIONAL COUPLING
Two or more polypeptides encoded by the same polycistronic mRNA can be translationally coupled. Two genes are translationally coupled if translation of the upstream gene affects the efficiency of the translation of the gene immediately downstream.
Figure 2.35 shows an example of how two genes could be translationally coupled. The TIR including the AUG initiation codon of the second gene is sequestered in a hairpin on the mRNA, so it cannot be recognized by an initiating ribosome. However, a ribosome arriving at the UAA stop codon for the first gene can open up this secondary structure, allowing another ribosome to bind to the downstream TIR and initiate translation of the second gene. Thus, translation of the second gene in the mRNA depends on the translation of the first gene. Mutations that disrupt translation of the upstream coding sequence (e.g., nonsense mutations or frameshift mutations that result in premature termination because the ribosome encounters nonsense codons in the new reading frame) therefore affect not only the gene in which they are located, but also the translationally coupled downstream gene.
POLAR EFFECTS ON GENE EXPRESSION
Some mutations that affect the expression of a gene in a polycistronic mRNA can have secondary effects on the transcription of downstream genes. Such mutations are said to exert a polar effect on gene expression. Several types of mutations can result in polar effects. One type of mutation that can cause a polar effect is an insertion mutation that carries a factor-independent transcriptional terminator. For example, if a transposon “hops” into a polycistronic transcription unit, the transcriptional terminators on the transposon may prevent the transcription of genes downstream of the insertion site in the same polycistronic transcription unit. Likewise, a “knockout” of a gene by insertion of an antibiotic resistance gene with a transcriptional terminator causes a polar effect on the genes downstream in the same transcription unit.
A second way a mutation in an upstream coding sequence can affect transcription of a downstream coding sequence is through effects on ρ-dependent termination of transcription. Recall that translation of mRNAs in bacteria normally occurs simultaneously with transcription and that the mRNA is translated in the same 5′-to-3′ direction as it is synthesized. Moreover, ribosomes often load onto a TIR as soon as it is vacated by the preceding ribosome, so that the mRNA is coated with translating ribosomes. If a nonsense mutation causes premature dissociation of ribosomes, the abnormally naked mRNA downstream of the mutation may be targeted by the transcription termination factor ρ, which may find an exposed rut sequence in the mRNA and cause transcription termination, as shown in Figures 2.15 and 2.36. The nonsense mutation therefore prevents the expression of the downstream gene by preventing its transcription. Such ρ-dependent polarity effects occur only if a rut sequence recognizable by ρ and a ρ-dependent terminator lie between the point of the mutation and the next downstream TIR.
Figure 2.35 Model for translational coupling in a polycistronic mRNA. (A) The secondary structure of the RNA sequesters the translational initiation region (TIR) of the second coding sequence (Gene 2) and blocks translation initiation (note that the Shine-Dalgarno shown is not the complete consensus sequence). (B) Translation of the first coding sequence (Gene 1) results in disruption of the secondary structure, allowing a ribosome to access the TIR of Gene 2 to translate the second coding sequence.
Superficially, translational coupling and polarity due to transcription termination have similar effects; in both cases, blocking the translation of one coding sequence affects the synthesis of another polypeptide encoded downstream on the same mRNA. However, the molecular bases of the two phenomena are completely different.
Protein Folding and Degradation
Translating the information in an mRNA into a polypeptide chain is only the first step in making an active protein. To be active, the polypeptide must fold into its final conformation. This is the most stable state of the protein and is determined by the primary structure of its polypeptides. Whereas some proteins fold efficiently into their active states, other proteins may need the assistance of other factors to increase the rate of folding into the active state and to prevent misfolding into an inactive state.
Protein Chaperones
Proteins called chaperones help other proteins fold into their final conformations. Some chaperones are dedicated to the folding of only one other protein, while others are general chaperones that help many different proteins to fold. We discuss only general chaperones here.
THE DnaK PROTEIN AND OTHER Hsp70 CHAPERONES
The Hsp70 family of chaperones is the most prevalent and ubiquitous type of general chaperone, existing in all types of cells with