Coupling Translation to Termination
The location of the leader peptide with respect to the four different regions capable of hybrid formation in the trp leader region provides a simple mechanism for regulating termination. If the 3-4 hybrid forms during transcription of this region, termination is possible because this hybrid is the “termination” loop. Conversely, if the 2-3 hybrid forms during transcription of leader, then formation of the “termination” loop is prevented. Finally, if the 1-2 hybrid forms, then region 2 is not available for formation of the 2-3 hybrid, but as RNA polymerase transcribes regions 3 and 4, they are free to base pair, and transcription terminates.
How can the presence or absence of tryptophan affect formation of the 1-2 or 2-3 hybrid? Ribosomes translating the leader region in the absence of charged tRNAs will stall at the trp codons and, owing to their location, they will block the formation of the 1-2 hybrid (Fig. 13.12). Formation of the 2-3 hybrid is not blocked, and as soon as these regions of the mRNA are synthesized, this hybrid forms. Consequently, the 3-4 hybrid does not form in time to terminate transcription, and termina-tion does not occur.
In the absence of any protein synthesis, ribosomes cannot bind to the leader region, and the 1-2 hybrid forms. In turn, this permits formation of the 3-4 hybrid. Termination follows. This happens in an in vitro transcription system, which, as we saw above, terminates transcription with a high probability at the attenuation site.
Figure 13.12 Possible structures of the leader mRNA in the presence of ribo-somes under the three conditions: tryptophan starvation, no protein synthesis, and tryptophan excess.
Finally, what happens in the presence of excess tryptophan? In cells containing adequate levels of tryptophan, much initiation, of course, will be blocked by the trp repressor. The transcription that is initiated however, will be largely terminated, for ribosomes either complete translation of the leader peptide and permit hybrids 1-2 and 3-4 to form, or ribosomes remain awhile at the termination codon. From this posi - tion they block formation of the 1-2 hybrid. In either case, the 3-4 hybrids form, and termination at the attenuator occurs.
It is necessary to note that the relative thermodynamic stability of the various leader hybrids is not important to attenuation. The factor determining whether termination will occur is which structures are not blocked from forming. The kinetics of formation of the base-paired structures should be on time scales less than milliseconds so that if they are not blocked from forming, they should form while the RNA is being synthesized. Then several seconds after initiating transcription, RNA polymerase reaches the attenuation site and terminates or not depend-ing on whether or not the 3-4 hybrid has formed. The interval between initiation and termination is much shorter than the interconversion time of many hybrid structures, and they therefore will not necessarily have had time to adopt their lowest-energy conformation (Table 13.1). The factor determining termination is which of the hybrid structures exists at the time RNA polymerase transcribes past the potential termi-nation site.
How does the system ensure that ribosomes initiate translation as soon as the leader has been synthesized? If ribosomes do not promptly initiate translation of the leader, premature termination at the attenu-ator will result. At the typical rates of ribosome binding to messenger, transcription could easily extend beyond the termination site before the first ribosome had a chance to bind to messenger and affect loop formation. The problem of forcing a ribosome onto the mRNA just as it emerges from the polymerase is solved in a simple way. The trp leader sequence possesses regions at which transcription is slowed due to pausing by polymerase. Most likely these pauses result from hairpin structures in the newly synthesized RNA. The duration of the pauses could be random, but if the average is long enough, most messengers could then have a ribosome bind and initiate translation. When polym-erase resumes transcription, the ribosome following immediately be-hind is properly situated to regulate attenuation.