Determination of the 5'-Ends of the 2.6...
The 5'termini of the two mRNAs were mapped by S-1 protection and primer extension analysis.Several different single-stranded DNA fragments with a 5'-labeled end within the puf B or A structural gene and a 3'-end upstream of the puf B structural gene were chosen as probes for hybridization with bulk RNA.The results are shown in Figure 5.The 32P-labeled single-stranded AccI（see Figure 1，fragment d，350 bp），AluI（see Figure 1，fragment a，500 bp），and Bst NI（see Figure 1，fragment e，190 bp）fragments were hybridized with RNA isolated from high light-grown cells and treated with S-1 nuclease.In all cases two distinct S-1-protected DNA fragments were observed in addition to the uppermost renatured DNA probe band.The sizes of the three pairs of S-1-protected fragments were 390 and 360 bp［see Figure 5（a）］，180 and 150 bp［see Figure 5（b）］，and 120 and 90 bp［see Figure 5（c）a］，respectively，when using the AluI，AccI，and BstNI restriction fragments.The larger S-1-protected fragments were always in excess of the smaller fragments（approximately（12—15）∶1），and the difference in sizes between the protected fragments was always approximately 30 bp.When S-1 protection experiments were performed on bulk RNA derived from cells undergoing a photosynthetic to aerobic shift it is clear that the larger fragment is more stable than the smaller one［see Figure 5（c）b and Figure 6（a）］with the ratio of the large to small S-1-protected fragment at time zero being 17∶1，at 5 min following the shift to aerobic conditions the ratio of the larger fragment to the smaller fragment was 20∶1；at 15 min，30∶1；and at 60 min，35∶1.Based on the fact the three pairs of protected fragments（e.g.larger fragments corresponding to 390，180，and 120 bp versus smaller ones corresponding to 360，150，and 90 bp）identified in S-l protection analysis share common properties（i.e.similar relative ratios and stabilities）to the two transcripts of the puf operon（0.5 and 2.6 kb）identified in Northern blots，these results map the 5'-termini of the small（0.5 kb）and large（2.6 kb）transcripts to approximately 100 and 70 nucleotides upstream of the start codon for the pu f B structural gene，respectively.
Figure 5 Analysis of the 5'-end of the two LH-Ⅰ transcripts of R.sphaeroides（pUI1A2）by S-1 mapping.Purified AluI（see Figure 1，fragment a），AccI（see Figure 1，fragment d），and BstNI（see Figure 1，fragment e）restriction fragments were 5'-end labeled with［γ-32P］ATP and T4polynucleotide kinase.The coding strand of the DNA was isolated，hybridized with RNA from photosynthetically［（a）、（b）、（c）a］or aerobically（c）b grown cells，treated with S-1 nuclease，and the S-1-protected fragments were electrophoresed on 5%polyacrylamide gels，as described under“Experimental Procedures”.The fragments of 500，350，and 190 bp in length represent renatured AluI，AccI，and BstNI DNA fragments，respectively.In addition，two S-1-protected fragments of different molar abundance（390，360 for AluI；180，150 for Accl；120，90 for BstNI）were observed in all three S-1 analyses.Based on the known DNA sequence for the puf operon，the 5'-ends of each transcript can be mapped to approximately 100 and 70 nucleotides upstream of the start codon of the pufB gene from this analysis
A 212-bp Accl-AluI restriction fragment（see Figure 1，fragment f）within the puf B and-A structural genes was used to identify the 5'-end of the puf operon transcripts by primer extension analysis.After labeling the 5'-end of this fragment with［γ-32P］ATP，the single-stranded DNA was separated on a 5%polyacrylamide gel and hybridized with RNA isolated from cells grown either under steady-state photosynthetic conditions or from photosynthetic cells following a shift to aerobic conditions for 1 h.Two primer-extended fragments of approximately 380 and 350 bp were observed either on 5%polyacrylamide gels［see Figure 6（b），a and b］or on 6%polyacrylamide，8.3 mol/L urea gels（data not shown）when using RNA from either culture.The sizes of these two primer-extended fragments were as expected from the results of S-1 mapping，so that the results of both the S-1 and primer extension analyses mapped the 5'-termini for the small and large puf operon transcripts to approximately 100 and 70 nucleotides upstream of the start of the puf B structural gene，respectively.
Figure 6 Analysis of the 5'-ends of the two transcripts from photosynthetic cells of R.sphaeroides（pUI1A2）after a shift to aerobic conditions by S-1 protection（a）and primer extension（b）assays.The Slanalysis using an AccI restriction fragment（see Figure 1，fragment d）in A was the same as shown in Figure 5.The RNA was isolated from cells exposed to high oxygen at different time intervals（0，5，15，60 min as represented by（a）a，（a）b，（a）c，（a）d）as in shown Figure 4.A more abundant and relatively stable band（180 nucleotides）and a less abundant and relatively unstable band（150 nucleotides）were observed on 5%polyacrylamide gels.The primer extension was performed using a AccI-AluI fragment（see Figure 1，fragment f）as a primer as described under“Experimental Procedures”.The extended cDNA using RNA isolated from photosynthetic cells（0 min）（（b）a）and from cells 60 min after exposure to oxygen（（b）b）were 380 and 350 nucleotides in length.The band at 210 nucleotides in length was renatured primer itself
In order to more precisely determine the 5'-ends for the puf operon transcripts，the 5-1-protected fragments obtained using the AccI restriction fragment（see Figure 1，fragment d）were analyzed on a 6%polyacrylamide，8.3 mol/L urea gel alongside a Maxam and Gilbert sequence ladder of the AtuI-AccI restriction fragment（see Figure 1，fragment g）.The restriction fragment used for Maxam and Gilbert sequencing was obtained by labeling the 5'-ends of the AccI restriction fragment（see Figure 1，fragment d）followed by digestion with AluI and purification of the large labeled AluI-AccI restriction fragment from a 5%polyacrylamide gel.Figure 7 shows the results of this analysis with the two 174-and 144-base pair S-1-protected fragments analyzed alongside the sequenced restriction fragment.This experiment，when analyzed together with the known DNA sequence of this region，［1］precisely maps the 5'termini for the small and large puf operon transcripts to 104 and 75 nucleotides upstream of the start of the pu f B structural gene.
Figure 7 Alignment of the 5'-ends of the puf operon transcripts with the DNA sequence of this region.Shown are the S-1-protected fragments obtained when RNA from steady-state photosynthetic cells（lane 1）or photosynthetic cells 1 h after a shift to aerobic conditions（lane 2）were hybridized to an end-labeled AccI single-stranded restriction fragment（see Figure 1，fragment d）prior to S-lnuclease treatment as described under“Experimental Procedures”.These samples were analyzed on a 6%polyacrylamide，8.3 mol/L urea gel alongside a Maxam and Gilbert sequence ladder containing the noncoding strand of the AluI-AccI restriction fragment（see Figure 1，fragment g）generated as described under“Results”
Since the DNA sequence of this region of the puf operon has been obtained by independent methods［1］we have shown only the G plus A and C plus T chemical reactions which allow positioning of the transcription initiation sites to 104 and 75 nucleotides upstream of the start of the pufB structural gene as shown.The corresponding DNA sequence of the noncoding strand is illustrated on the left with the transcript initiation sites highlighted with a circle or a box.The experiments performed to date have not allowed precise resolution as to which of the two possible A residues initiates transcription for the large transcript，so both nucleotides are highlighted.However，for the purposes of further discussion and clarity in the text we have assumed it to be the second base which is 104 nucleotides upstream of the initiation of the pufB structural gene
