Differential Decay of the Large and Small puf Oper...

Differential Decay of the Large and Small puf Operon Transcripts

Ⅰn order to obtain a quantitative estimate of the kinetics of decay of the 2.6-and 0.5-kb mRNA species，the decay rates of these transcipts in high light-grown cells of R.sphaeroides 2.4.1（pUI1A2）were studied following inhibition of transcription using rifmpicin.Samples were taken at various time intervals for the assay of specific mRNA levels by dot blots and Northern hybridizations employing the specific prodes described above and in Figure 2.The results as shown in Figure 3（a）where it can be seen that the decay of these transcripts followed exponential kinetics after the addition of rifampicin.The half-life（t1/2）of the mRNA sequences specifying the LH-1 structural genes was 20 min，whereas the corresponding value for the sequences corresponding to pufL and pufM as 9 min.Similar decay rates for the individual mRNA sequences requences by the two major transcripts were also obtained by analysis of Northern hybridizations（data not shown）.Because the LH-Ⅰ-sequences contained within the small transcript are approximately 15-fold more abundant than homologous sequences encoded on the large transcript in high light-grown cells（see above），the values reported for the half-life of the LH-Ⅰ specific sequence have not been corrected for the decay of the large transcript.However，such a correction would be minimal and would not significantly alter the calculated results for mRNA half-lives.

The kinetics of decay of these transcripts were different when the high light-grown cells were shifted to darkness.The levesls of the small and large transcripts specific for LH-Ⅰ，RC-L，and RC-M polypeptides increased for the first 5 min following a shift to the dark and then decreased at different rates.In general，the turnover of these transcripts was slower under such conditions relative to the rifampicin-treated cells［see Figure 3（c）］.The initial increase in transcription of this operon may reflect a derepression in the rate of transcription of this operon due to light limitation，but it is clear that the small transcript is more stable than the large transcript under these conditions.Because of the physiologic complexity of the light to dark shift we cannot draw any further major conclusions.

Following a shift from photoheterotrophic to aerobic conditions the half-lives of the transcripts（as determined by dot hybridization）homologous to the LH-Ⅰ，RC-L，and RCM.Similar results were also obtained from Northern hybridization analysis（see Figure 4）.It can also be seen that the large（2.6 kb）transcript was rapidly degraded after exposure to O2 and was present only at very low levels even after 10 min［see Figure 4（b）and（c）］.In contrast the level of the small transcript decreased more slowly and was clearly detectable 30 min following the shift to aerobic conditions［see Figure 4（a）］.An additional important feature of the results of the Northern hybridization was that significant amounts of discretesized transcripts（2.0，1.8，1.4，and 1.1 kb）were observed when the X-ray films were exposed for long periods［see Figure 4（b）and（c）］.All of these intrermediate-sized transcripts hybridized only with probes specific of the RC-L and RC-M structural genes.Further，when the specific RC-L and RC-M probes were used in Northern hybridization［see Figure 4（b）and（c）］many other hybridizing species，resulting in a smear，were observed between the 2.6-and 1.1-kb regions.One possible interpretation of these resulted from processing of the large transcipt at specific sites located toward the 5'-end of the transcript；the smear may be caused by extensive degradation of the large transcript，and the gaps between the individual degradation products may represent some RNA sequences which were relatively resistant to ribonuclease attack.On the other hand，when the LH-Ⅰ sepecific probe was employed［see Figure 4（a）］the loss of the large transcript appeared to be more rapid，and fewer intermediates were apparent.These data coupled with the kinetic data in Figure 3 and the previously discussed data in Figure 2 suggest that the breakdown of the large transcript proceeds from the 5'-to the 3'-end of the mRNA.