ANALYTICAL BIOCHEMISTRY 119, 86-89 (1982)

Sequencing Psoralen Photochemically Reactive Sites in Escherichia coli 16 S rRNA

DOUGLAS C. YOUVAN* AND JOHN E. HEARST?,’

*Department of Biophysics and Medical Physics and tDeparrment of Chemistry, University of California. Berkeley, California 94720

Received February 24, 198 1

The photochemical modification of 16 S rRNA by longwave ultraviolet light in the presence of hydroxymethyltrimethyl psoralen (HMT) produces uridine-psoralen adducts. Psoralen- modified 16 S rRNA is primed with a 3’-proximal restriction fragment and copied with reverse transcriptase. The average length of the cDNA transcripts decreases as the number of co- valently bound psoralen adducts on the template RNA increases. Adducts stop reverse tran- scriptase and abbreviated cDNAs terminate at the positions of the lesions. The cDNA reverse transcripts of psoralen-reacted 16 S rRNA are electrophoresed in parallel with a dideoxy sequence (Sanger method) which uses the same restriction fragment as a primer. The psoralen- induced cDNA stops are found to be highly localized hot spots. In the 1 SO-nucleotide sequence investigated at sequence resolution, two hot spots for psoralen reaction are found. In both cases the psoralen-induced stops are at UpU sequences near the bases of predicted hairpins.

The kinetics of polymerization of nucleic acid by various cellular polymerases is re- sponsive to structural features in the tem- plate. Attenuation of the rate of polymer- ization on single-stranded templates in vitro can be reduced to simpler biophysical phe- nomena due to the primary and secondary structures of the template (l-6).

The kinetics of reverse transcription on ribosomal RNA (rRNA) templates is atten- uated by natural and chemical base modi- fications. The rate-limiting or blocked step in complementary DNA (cDNA) synthesis by AMV reverse transcriptase (avian my- eloblastosis virus RNA-directed DNA nu- cleotidyltransferase; deoxynucleosidetri- phosphate:DNA deoxynucleotidyltrans- ferase, EC 2.7.7.7)* occurs at the positions of base modifications in the template RNA.

’ To whom reprint requests should be addressed. * Abbreviations used: AMV reverse transcriptase,

avian myeloblastosis virus RNA-directed DNA nucleo- tidyltransferase; m*G, N*-methylguanine, am4, 3-(3- amino-3-carboxypropyl)- 1 -methylpseudouridine; mz6A,

N* -Methylguanine (m*G) causes a 3-min pause in cDNA elongation on Escherichia coli 16 S rRNA ( 1). The hypermodified nu- cleoside 3-( 3-amino-3-carboxypropyl)- l- methylpseudouridine (am%) causes an ab- solute stop in cDNA elongation on Dro- sophila melanogaster 18 S rRNA (5). The sequence m26Am26A stops reverse transcrip- tase on the 3’-end of small subunit ribosomal rRNAs (4). If the template base modifica- tion affects base-pairing, then the rate of cDNA elongation is attenuated.

In this communication, it will be shown that hydroxymethyltrimethyl psoralen (HMT) adducts produced by uv irradiation (7,8) of template RNA produces reverse- transcriptase stops. These adducts are highly localized in the template and react only with a small number of uridine residues. The

N6,N6-dimethyladenine; HMT, hydroxymethyltrime- thy1 psoralen; MgOAc, magnesium acetate; ddNTP, dideoxyribonucleoside triphosphate; UpU, Uridyl-Uri- dine.

0003-2697/82/010086-04$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of rsprcduction in any form reserved.

86

PSORALEN PHOTOCHEMICALLY REACTIVE SITES IN 16 S rRNA 87

0

FIG. 1. One possible stereoisomer of the HMT-uri- dine photochemical monoadduct.

psoralen-induced stops are doublets and have been observed in HMT-reacted RSV (rous sarcoma virus) RNA (Swanstrom, R., personal communication). The structure of a psoralen-uridine adduct is shown in Fig. 1. Although the drug does not react directly with a base-pairing ligand, base-pairing with monomer dATP is probably blocked steri- tally. This putative stereoisomer is analo- gous to the structure of an HMT-thymidine monoadduct recently found in DNA (9).

MATERIALS AND METHODS

HMT-photoreacted RNA. HMT-cross- linked E, coli 16 S rRNA was a gift from Paul Wollenzien. These samples are identi- cal to those used in the electron microscopic determination of long-range loop interaction frequency as a function of the number of HMTs incorporated per molecule ( 10). Samples were irradiated 10 min by 100 mW/cm’ 360-nm uv light in 5 mM MgOAc, 30 InM Tris-HCl (pH 7.6), and 0.33 M NH&l. The concentration of tritium-la- beled HMT in the irradiation mixes was adjusted to yield RNA samples with ap- proximately 2 or 8 psoralensj 16 S rRNA molecule.

Primers, reverse transcription, and se- quencing. Priming of 16 S rRNA with the HinfI restriction fragment, kinetic analysis of cDNA elongation on alkaline agarose gels, and dideoxy sequencing of the psoralen stops were identical to the experimental con- ditions previously reported ( 1).

RESULTS

Escherichia coli 16 S rRNA with 2 or 8 HMTs/ 16 S rRNA molecule was primed with the 3’-proximal HinfI 7 S restriction fragment (1 ,l 1) and copied with AMV re- verse transcriptase. An autoradiogram of this cDNA electrophoresed on an alkaline agarose gel is shown in Fig. 2. Templates not reacted with psoralen were transcribed 20 or 40 min and the templates with 2 or 8 psoralens were transcribed 40 min. Com- pared to native 16 S rRNA, the amount of full-length cDNA transcribed on templates averaging 2 HMTs/l6 S rRNA molecule is greatly reduced and the distribution of cDNA lengths is shifted toward lower molecular weights. No full-length transcript is detected on templates averaging 8 psoralens/mole- cule and the average length of cDNA is re- duced even more. Since the psoralen-RNA irradiation is done at 360 nm, we conclude that the only modifications made in the 16 S rRNA are the psoralen adducts and that these adducts inhibit the elongation of cDNA by AMV reverse transcriptase.

Complementary DNAs synthesized by AMV reverse transcriptase were electropho- resed on lanes parallel to a dideoxy nucleo-

minutes 20 40 40 40 PSOWlenS 0 0 2 8 EA

FIG. 2. Autoradiogram of a 1.4% alkaline agarose gel showing the decreasing lengths of cDNA synthesized on templates with increasing amounts of HMT per 16 S rRNA. The total time of reverse transcription and the average number of HMTs per 16 S rRNA molecule are indicated above each lane, P, primer; E, transcript ex- tending to the S-terminus; 1, m% at 1206; 2, m*G at 965. Band 3, a psoralen-induced stop. Marker (M) lane contains Hind111 SV40 fragments: 216, 448, 527, 1102, 1170, and 1769 nucleotides.

88 YOUVAN AND HEARST

tide terminated sequence (Fig. 3). Since the psoralen-terminated cDNA and the dideoxy- terminated cDNA start from the same re- striction fragment primer, the psoralen stops can be positioned exactly in reference to the dideoxy sequence. Two very discrete psora- len-induced stops are observed at sequence positions 1306- 1307 and 12 lo- 12 11. Posi- tioning of these stops is facilitated by the preferential reaction of psoralen with uri- dines (12). The entire sequence of E. coli 16 S rRNA has been determined ( 11). Psora- len-induced band 3 is very close to the 3’-end of the primer and is observed in the low- resolution alkaline agarose gel (Fig. 2). This band is a doublet on the sequencing gel and aligns with the UpU sequence at 1306- 1307. The only other UpU sequence within se- quence resolution of the Hinfl primer is at sequence position 1210-1211 which is the second psoralen-induced stop.

DISCUSSION

Two psoralen-induced stops in cDNA elongation on HMT photochemically re- acted 16 S rRNA have been localized at sequence positions 1210-1211 and 1306- 1307 (numbered from the j/-terminal RNA base). These are the only two psoralen hot spots found between positions 1340 and 1200. In addition, they are the only two UpU sequences in this region of the rRNA. An- other similarity exists between these two sites; both sites are at the bases of predicted ( 13) hairpin structures (see Fig. 4). Finding other examples of psoralen-reactive sites should be useful in discriminating between the reactivity toward particular sequences and toward particular types of secondary structures, i.e., a nonreactive UpU sequence would rule out a preferential reaction with this dimer in favor of a reaction with a hair- pin stem.

With the current advances being made in oligonucleotide synthesis, it should become increasingly expedient to synthesize a set of

12l4

190s

1.m

1262

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FIG. 3. Autoradiogram of an 8% dideoxy-sequencing gel used to position HMT induced stops in the 16 S rRNA sequence lanes: U, G, C, and A terminated by the complementary ddNTP, Y and Z, cDNAs synthe- sized on native template with no ddNTPs; and W and X, cDNAs synthesized on templates with 2 and 8 pso- ralens/l6 S rRNA molecule with no ddNTPs. Band 3, HMT stop at 1306-1307; 4, HMT stop at 1210-1211; 5 and 6, HMT-induced stops beyond sequence resolu- tion; and 2, the second m2G pause at sequence position 965.

primers for E. coli 16 S rRNA. The synthe- sis of milligram amounts of 15-nucleotide- long oligomers, which are complementary to

PSORMEN PHoTocHJ5MKALLY REACTIVE !SlTES M 16 S rRNA 89

AA c c

6 u u c

t-6 iI4

1310 - :i 6-C 6-C

66AUU-AU6AA

+ Lo

C U A

6 A

;:; 6-C U-A - 1200 A-U

; 6-c$

5 f 6-C

lla0 - 6-C A-U

A 6 6 A-U ‘+ AC

F~~4.Haiqims(l3)i~tkvkimityaftktwopmr- fabastk upu rcqaaDa (8mws) at 1210-1211 amd 13061307.

tbeRNAeveryXlOn~wouldform tkbasisofanassaysystemcapabIeofle caking cbemid adduas to sapence res- oiuth over the entire sequence.

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