Other Bacterial Operons
Page 813 Figure 19.12 Leader peptide sequences specified by biosynthetic operons of E. coli. All contain multiple copies of amino acid(s) synthesized by enzymes coded for by operon. and render it less stable. Some mutations, however, increase termination at the attenuator. One of these interferes with base pairing between regions 2 and 3, allowing region 3 to be available for pairing with region 4 even when region 1 is bound to a stalled ribosome. Another mutation occurs in the AUG initiator codon for the leader peptide so that the ribosome cannot begin its synthesis. Transcription Attenuation Is a Mechanism of Control in Operons for Amino Acid Biosynthesis Attenuation is a common phenomenon in bacterial gene expression; it occurs in at least six other operons that code for enzymes catalyzing amino acid biosynthetic pathways. Figure 19.12 shows the corresponding leader peptide sequences specified by each of these operons. In each case, the leader peptide contains several codons for the amino acid product of the biosynthetic pathway. The most extreme case is the 16residue leader peptide of the histidine operon that contains seven contiguous histidines. Starvation for histidine results in a decrease in the amount of histidinyltRNAHis and a dramatic increase in transcription of the his operon. As with the trp operon, this effect is diminished by mutations that interfere with the level of charged histidinyltRNAHis. Furthermore, the nucleotide sequence of the attenuator region suggests that ribosome stalling at the histidine codons also influences the formation of alternate hairpin loops, one of which resembles a termination hairpin followed by several U residues. In contrast to the trp operon, transcription of the his operon is regulated entirely by attenuation; it does not possess an operator that is recognized by a repressor protein. Instead, the ribosome acts rather like a positive regulator protein, similar to the cAMP–CAP complex discussed with the lac operon. If the ribosome is bound to (i.e., stalled at) the attenuator site, then transcription of the downstream structural genes is enhanced. If the ribosome is not bound, then transcription of these genes is greatly reduced. Transcription of the other operons shown in Figure 19.12 can be attenuated by more than one amino acid. For example, the thr operon is attenuated by either threonine or isoleucine; the ilv operon is attenuated by leucine, valine, or isoleucine. This effect can be explained in each case by stalling of the ribosome at the corresponding codon, which, in turn, interferes with the formation of a termination hairpin. Although not proved, it is possible that in the cases of the longer leader peptides, stalling at more than one codon is necessary to achieve maximal transcription through the attenuation region. 19.5— Other Bacterial Operons Synthesis of Ribosomal Proteins Is Regulated in a Coordinated Manner Many other bacterial operons have been studied and found to possess the same general regulatory mechanisms as the lac, trp, and his operons, as discussed Page 814 Operon Regulator protein Proteins specified by the operon Spc S8 L14L24L5S14S8L6L18S5L15L30 S10 L4 S10L3L2L4L23S19L22S3S17L16L29 str S7 S12S7EF•GEF•Tu a S4 S13S11S4aL17 L11 L1 L11L1 rif L10 L10L7b Figure 19.13 Operons containing genes for ribosomal proteins E. coli. Genes for the protein components of the small (S) and large (L) ribosomal subunits of E. coli are clustered on several operons. Some of these operons also contain genes for RNA polymerase subunits a, b, and , and protein synthesis factors EF∙G and EF∙Tu. At least one of the protein products of each operon usually regulates expression of that operon (see text). in Section 19.4. However, each operon has evolved its own distinctive quirks. For example, one interesting group of operons are those containing the structural genes for the 70 or more proteins that comprise the ribosome (Figure 19.13). Each ribosome contains one copy of each ribosomal protein (except for protein L7L12, which is probably present in four copies). Therefore all 70 proteins are required in equimolar amounts, and it makes sense that their synthesis is regulated in a coordinated fashion. Characterization of this set of operons is not yet complete, but six operons, containing about onehalf of the ribosomal protein genes, occur in two major gene clusters. One cluster contains four adjacent operons (str, Spc, S10, and a), and the other two operons are near each other elsewhere in the E. coli chromosome. There is no obvious pattern to distribution of these genes among different operons. Some operons contain genes for proteins of just one ribosomal subunit; others code for proteins of both subunits. In addition to structural genes for ribosomal proteins, these operons also contain genes for other (related) proteins. For example, str operon contains genes for the two soluble translation elongation factors, EF∙Tu and EF∙G, as well as genes for some proteins in the 30S ribosomal subunit. The a operon has genes for proteins of both 30S and 50S ribosomal subunits plus a gene for one of the subunits of RNA polymerase. The rif operon has genes for two other protein subunits of RNA polymerase and genes for ribosomal proteins. A common theme among the six ribosomal operons is that their expression is regulated by one of their own structural gene products; that is, they are selfregulated. The precise mechanism of this selfregulation varies considerably with each operon and is not yet understood in detail. However, in some cases the regulation occurs at the level of translation, not transcription as discussed for the lac and trp operons. After the polycistronic mRNA is made, the "regulatory" ribosomal protein binds to this mRNA and determines which regions, if any, are translated. In general, the ribosomal protein that regulates expression of its own operon, or part of its own operon, is a protein that is associated with one of the ribosomal RNAs (rRNAs) in the intact ribosome. This ribosomal protein has a high affinity for the rRNA and a lower affinity for one or more regions of its own mRNA. Therefore a competition between the rRNA and the operon's mRNA for binding with the ribosomal protein occurs. As the ribosomal protein accumulates to a higher level than the free rRNA, it binds to its own mRNA and prevents the initiation of protein synthesis at one or more of the coding sequences on this mRNA (Figure 19.14). As more ribosomes are formed, the excess of this particular ribosomal protein is used up and translation of its coding sequence on the mRNA can begin again. Page 815 Figure 19.14 Selfregulation of ribosomal protein synthesis. If free rRNA is not available for assembly of new ribosomal subunits, individual ribosomal proteins bind to polycistronic mRNA from their own operon, blocking further translation. Stringent Response Controls Synthesis of rRNAs and tRNAs Bacteria have several ways in which to respond molecularly to emergency situations; that is, times of extreme general stress. One of these situations is when the bacterium does not have a sufficient pool of amino acids to maintain protein synthesis. Under these conditions the cell invokes what is called the stringent response, a mechanism that reduces the synthesis of the rRNAs and tRNAs about 20fold. This places many of the activities within the cell on hold until conditions improve. The mRNAs are less affected, but there is also about a threefold decrease in their synthesis. Figure 19.15 Stringent control of protein synthesis in E. coli. During extreme amino acid starvation, an uncharged tRNA in the A site of the ribosome activates the relA protein to synthesize ppGpp and pppGpp, which, in turn, are involved in decreasing transcription of the genes coding for rRNAs and tRNAs. The stringent response is triggered by the presence of an uncharged tRNA in the A site of the ribosome. This occurs when the concentration of the corresponding charged tRNA is very low. The first result, of course, is that further peptide elongation by the ribosome stops. This event causes a protein called the stringent factor, the product of the relA gene, to synthesize guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), from ATP and GTP or GDP as shown in Figure 19.15. Stringent factor is loosely associated with a few, but not all, ribosomes of the cell. Perhaps a conformational change in the ribosome is induced by occupation of the A site by an uncharged tRNA, which, in turn, activates the associated stringent factor. The exact functions of ppGpp and pppGpp are unknown. However, they seem to inhibit transcription initiation of the rRNA and tRNA genes. In addition they affect transcription of some operons more than others.