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FIGURE 16-21 Transcription termination at the trp attenuator. How transcription termination at the trp operon attenuator is controlled by the availability of tryptophan. Ir, (a) (conditions of high tryptophan), sequence 3 can pair with sequence 4 to form the transcription teiminaton hairpin. In (b) (conditions of low tryptophan), the nbosome stalls at ad|acent tryptophan codons, leaving sequence 2 free to pair with sequence 3. thereby preventing formation of the 3, 4, termination hairpin. In (c) (no protein synthesis), if no nbosome begins translation of the leader peptide AUG. the hairpin forms by painng of sequences 1 and 2, preventing formation of the 2, 3, hairpin, and allowing formation of the hairpin at sequences 3,4. The trp enzymes are not expressed.

involves recognition of specific secondary structures in the mRNA. We consider here the regulation of the genes that encode ribosomal proteins.

Correct expression of ribosomal protein genes poses an interesting regulatory problem for the cell. Each ribosome contains some 50 distinct proteins that must be made at the same rate. Furthermore, the rate at which a cell makes protein, and thus the number of ribosomes it needs, is tied closely to the cell's growth rate; a change in growth conditions quickly leads to an increase or decrease in the rate of synthesis of all ribosomal components. How is all this coordinated regulation accomplished?

Control of ribosomal protein genes is simplified by their organization into several opérons, each containing genes for up to 11 ribosomal proteins (Figure 16-22). The genes for some nonribosomal proteins whose synthesis is also linked to growth rate are contained in these opérons, including those for RNA polymerase subunits a, (i, and As with other opérons, these opérons are sometimes regulated al the level of RNA synthesis. But, the primary control of ribosomal

b low tryptophan leader tryptophan codons protein synthesis is at the level of translation of the mRNA, no! transcription. The following simple experiment shows the distinction

When extra nopies of a ribosomal protein operon are introduced into the cell, the amount of mRNA increases correspondingly, but synthesis of the proteins stays nearly the same. Thus, the cell compensates for extra mRNA by curtailing its activity as a template. This happens because ribosomal proteins are repressors of their own translation.

For each operon, one (or a complex of two) ribosomal proteins hinds die messenger near the translation initiation sequence of one of the iirst genes in the operon, preventing ribosomes from binding and initiating translation. Repressing translation of the first gene also prevents expression of some or all of the rest. This strategy is very sensitive. A few unused molecules of protein L4, for example, will shut down synthesis of that protein, as well as synthesis of the other ten ribosomal proteins in its operon. In this way. these proteins are made just at the rate they are needed for assembly into ribosomes.

How one protein can function both as a ribosomal component and as a regulator of its own translation is shown by comparing the sites where that protein binds to ribosomal RNA and to its messenger RNA. These sites ate similar hoth in sequence and in secondary st rue luff

Box 16-4 Riboswrtches

Gene regulation typically involves regulatory proteins that control the expression of genes at the level cf transcription or translation. Not all gene expression is governed by regulatory proteins, however. The tryptophan operon of E colt, as we have seen, responds to the cellular levei of te end product (tryptophan) by an attenuation mechanism involving a leader RNA but no dedicated regulatory protein. Another example of gene regulation that does nut involve a regulatory protein is the ribosomal RNA (rRNA) genes of £?. to//, whose rate of transcription is strongly influenced by the growth rate of the cell.

It turns out that RNA polymerase forms unstable complexes at the promoters for rRNA genes, and these complexes are highly sensitive to the concentration of the nucleotide that initiates transcription (usually ATP). Hence, under conditions ot rapid growth when the cellular levels of ATP are high, the RNA polyrnerase-promoter complexes are productive, and the rRNA genes are transcribed at a high rate Conversely, under conditions of nutrient limitation when the growth rate and cellular ATP levels are low, tnitiatifjn by RNA polymerase is inefficient and rRIMA genes arc transcribed aî a low rate. This nudeotide-sensing system is perhaps the simplest of all transcriptional control mechanisms as it involves no regulatory proteins and is solely determined by the special properties of fRNA gene promoters.

Vet another example of gene regulation without regulatory proteins is the riboswitch. Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation. For example, many genes whose function is related to tine amino acid methionine in the bacterium Bacillus subtilis are controlled by a 200-nudeotide-iong, untranslated leader RNA that can adopt alternative structures: one involving a stem-loop transcription terminator and the other an antrterminator. S-adenosyl methionine, but not methionine itself (or other methtonine-related small molecules), binds TO these leader RNAs to stabilize the transcription termination structure. These leader RNAs are therefore switches (rtboswttches) that sense cellular levels ot S-adenosy! methionine and thereby control transcriptional read-through into the downstream gene. Many examples of riboswitches are now known, each responding to a different metabolite, such as vitamin B12, thiamine pyrophosphate, flavin mononucleotide, lysine, guanine, and adenine (Box 16-4 Figure l) Some riboswitches operate at the level of transcription termination but others operate at the level of translation, controlling the formation of an RNA structure that blocks binding of the nbosome to the mRNA for the downstream gene. Riboswitches are found not only in bacteria, but evidently also in archaca, fungi, and plants.

Another kind of riboswitch deserves special mention. Rather than responding to a metabolite, these leader RNAs respond to uncharged tRNA. Thus, certain genes, notably genes for aminoacyl tRNA synthetases {see Chapter 14), are controlled by a transcription termination mechanism that involves a 200-to 300-nucleotide long, untranslated, leader RNA that directly and specifically interacts wtth the cognate, uncharged tRNA for the synthetase. This interaction stabilizes the leader RNA in its antitermination structure so that transcription into the adjacent synthetase gene can proceed. Speoficrty is achieved in part by a "codon-anticodon" interaction between the tRNA and the leader RNA Because only uncharged tRNA can bind to the leader, transcriptional read-through is only stimulated when the cognate amino acid is in short supply and the level of uncharged tRNA in the cell nses.

&12 riboswitch coenzyme b12

FMN riboswitch flavin mononucleotide

lysine riboswitch

SAM ribo switch

S-aderiosyl-methionin«

TPP riboswitch thiamine pyfophosphate

guanine adenine riboswitch

BOX 16-4 FIGURE 1 Riboswitches participate in fundamental genetic control The secondary structures oí the seven known riboswitdtó and the metdbolites they sense are shown here. (Source: Adapted from Mandat M., Boesc 8., Bairick J.E., Winkler W.C., and Breaker R.R. 2003. Cell 113: 577 - 586; figure / Panel A, page 584. Copyright © 2000, with permission of Elsevier.)

510 Gene Regu/nijoH in Prokaryotes Box 16-4 (Continued)

&12 riboswitch coenzyme b12

FMN riboswitch flavin mononucleotide

SAM ribo switch

S-aderiosyl-methionin«

TPP riboswitch thiamine pyfophosphate

BOX 16-4 FIGURE 1 Riboswitches participate in fundamental genetic control The secondary structures oí the seven known riboswitdtó and the metdbolites they sense are shown here. (Source: Adapted from Mandat M., Boesc 8., Bairick J.E., Winkler W.C., and Breaker R.R. 2003. Cell 113: 577 - 586; figure / Panel A, page 584. Copyright © 2000, with permission of Elsevier.)

(Figure 16-23), The comparison suggests a precise mechanism of regulation. Since the binding site in the messenger includes the initiating AUG, niRNA bound by excess protein S8 (in this example) cannot attach to ribosomes to initiate translation. (This is analogous to Lac repressor binding to the lac promoter and thereby blocking access to RNA polymerase.) Binding is stronger to ribosomai RNA than to mRNA, so translation is repressed only when all need for the protein in ribosome assembly is satisfied.

guanine riboswitch

5"

guanine adenine

NH, adenine riboswitch

lysine riboswitch

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