Structural changes in the T4 gene 32 protein induced by DNA polynucleotides.

Alterations in the structure of the DNA-binding protein specified by gene 32 of bacteriophage T4 have been detected using partial trypsin digestion as a conformational probe. Limited tryptic hydrolysis of the gene 32 protein removes a fragment ("B" region), of 21 amino acids from the NH2 terminus and a 6,200-dalton fragment ("A" region) from the COOH terminus. Poly(dT), poly(dC), and single-stranded DNA increase the rate of tryptic hydrolysis of the "A" region but decrease the rate of tryptic hydrolysis of the "B" region. Oligonucleotides, which are too short to permit cooperative binding of the gene 32 protein, do not alter the rate of tryptic hydrolysis of either the "A" or "B" regions. A model which accounts for these findings requires that the "B" region be involved in gene 32 protein:gene 32 protein interactions when the gene 32 protein: DNA complex is formed. As a consequence of the gene 32 protein:DNA interaction, the "A" region should be able to participate more effectively in vivo and in vitro with other proteins involved in T4 DNA metabolism.

" Cysteine was quantitated as cysteic acid and methionine was quantitated as methionine sulfone after performic acid oxidation according to the procedure of Glazer et al. (20). Tryptophan was estimated spectrophotometrically.
gradient was spun at 40,000 rpm for 70 min in a SW 50.1 rotor which puts the peak of virus in the center of the gradient.
Fractions were collected from the gradient and the virus pelleted by centrifugation at 45,000 rpm in the SW 50.1 rotor for 3 h. The single-stranded fd DNA was isolated by the phenol extraction procedure developed by Marvin and Schaller (24). Synthetic poly-and oligonucleotides were obtained from P-L Biochemicals and were used without further purification.
The time course of the carboxypeptidase digestion on performic acid-oxidized 32P is shown in Fig. 1A Fig. 2B.
Dansyl-Edman degradation of the 28,800-dalton species gave the same NH,-terminal sequence as found in the native protein (Fig. 3). The time course of carboxypeptidase digestion is identical for both the 28,800-and 27,500-dalton fragments (Fig. lB) and is consistent with the COOH-terminal sequence: (Thr, Ala)-Ala-Lys-COOH. This differs from the COOH-terminal sequence of the native protein (Fig. 3) so it appears that both the 27,500-and 28,800-dalton fragments lack a 6,200 molecular weight piece which must be at the COOH terminus of the intact protein.
This region was referred to as the "A" peptide by Moise and Hosoda (14) and therefore we will refer to the 28,800-dalton fragment, which is identical with the native 32P except for lacking the COOH-terminal A region, as 32P*-A.
We believe that 32P*-A is the same as the P32*-I product previously described by Moise and Hosoda (14). The time course for the release of free amino acids shown above is consistent with a COOH terminus of (Thr, Ala)-Ala-Lys-COOH for both 32P*-A (0) and 32P*-(A + Bl (0). through 24 in the native protein (Fig. 3). Thus 32P*-(A + B) is missing the first 21 amino acids ("B" region) from the NH, terminus as well as the 6,200-dalton COOH-terminal "A" region. These are the only two regions that can be removed by mild proteolysis of the native protein. 32P*-(A + B) is probably identical with the 32P*-III species described by Moise and Hosoda (14). The amino acid composition of the two cleavage products, 32P*-A and 32P"-(A + B), is shown in Table I. As shown in this table the "A" region contains a high proportion of Asx residues, in agreement with Anderson and Coleman (25). In contrast to the "A" region, the "B" region contains a high proportion of lysine and arginine residues. Isoelectric focusing in polyacrylamide gels (data not shown) reveals that 32P*-(A + B) is more basic than 32P.  Fig. 4A) about 50% of the protein is still intact at the end of the digestion in the absence of DNA while the remainder is in the form of 32P*-A or 32P*-(A + B), the latter appearing as a shoulder on the 32P*-A peak. As shown in Scans b to d in the same figure, the addition of increasing amounts of single-stranded DNA results in an accelerated rate of removal of the "A" region. This effect is most pronounced when the base/gene 32 protein ratio is above 4.6 as shown by the lack of a 32P peak in Scan c (Fig. 4A). If the trypsin concentration is increased 32-fold, then in the course of the 60-min digestion, all of the native protein is coverted to 32P*-(A + B) as shown by the top scan in Fig. 4B. In contrast, if single-stranded DNA in a base132P ratio of 4.6 is included in an otherwise identical reaction mixture (Scan b, Fig. 4B) approximately 70% of the 32P retains the "B" region at the end of the digestion. Fig. 4B (Scans c and d) demonstrates that protection of the "B" region from trypsin digestion be- The trypsin concentration was 8.6 pg/ml throughout and the base ratio of fd DNA to 32P, in b, c, and d was 4.6, 9.2, and 18.4, respectively. comes less pronounced as the base/32P ratio is increased above 4.6. While this result was unexpected, it is consistent with the model presented in the discussion. The data in Fig. 4, A and B, is summarized in Table III and clearly shows that singlestranded DNA accelerates the rate of cleavage of the "A" region and depresses the rate of tryptic hydrolysis of the "B" region of 32P. Homopolynucleotides Except Those Containing Guanine Facilitate Trypsin Hydrolysis of "A" Region. Only Polydeoxypyrimidines Protect "B" Region against Trypsin Digestion -In view of the reported absence of base specificity in the binding of the gene 32 protein to DNA (261, it was assumed that all homopolynucleotides would have the same effect on the rate of trypsin digestion of 32P. As shown in Table IV, this was not the case. With the low concentration of trypsin used in the first experiment, 21% of the "A" region is removed in the absence of any added polynucleotide.
Homopolynucleotides, except those containing guanine, increase the rate of proteolysis of the "A" region by 3-to 4-fold as evidenced by the data in the second column of  and the "A" and "B" region release was quantitated by densitometric scanning of the SDS-polyacrylamide gels. "A" region release was measured after a 60-min incubation with 0.04 pg/ml of trypsin and "B" region release was determined after a similar incubation in the presence of 8.0 pg/ml of trypsin.
In those experiments with added homopolynucleotide, the base concentration was 56 /AM.

Polynucleotide
"A" region rel eased "B" region rel eased with low trypsin with high trypsin % % difference was observed between ribo-and deoxyribonucleotides. The same experiment was repeated in the presence of a high concentration of trypsin where all of the "A" and most of the B" region were removed during digestion. The last column in Table IV indicates that, in the absence of added polynucleotides, 94% of the gene 32 protein is converted to 32P*-(A + B). Of the homopolynucleotides tested, only poly(dT) and poly(dC) were able to reduce the rate of conversion of 32P*-A to 32P*-(A + B) and thus provide protection of the "B" region from tryptic cleavage. None of the polyribonucleotides, including poly(C), showed this effect.
The homopolynucleotides in Table IV  The trypsin concentration was 8.0 *g/ml throughout and the poly(dTY32P base ratio in each digest is indicated above. Samples were run on an SDS-polyacrylamide gel, and the area under the 32P*-A and 32P*-(A + B) peaks was determined by scanning the stained gel with a Joyce-Loebl densitometer.
The per cent protection of the 73" region is the ratio of the 32P*-A peak to the total peak area.
classes with respect to their effect on the partial tryptic hydrolysis of the gene 32 protein. Poly(dG) and poly(G) are unique in that they do not appear to affect the rate of tryptic cleavage of either the "A" or "B" regions of the protein. The simplest explanation for this result is that the stability of the tetra-stranded helices assumed by guanine-containing homopolynucleotides in solution (27) prevents poly(G) and poly(dG) from binding the gene 3.2 protein. Poly(dA), -(Cl, -(U), and -(A) all enhance the rate of trypsin digestion of the "A" region without affecting the rate of release of the "B" region. In contrast, poly(dT) and poly(dC) mimic the behavior of fd DNA (Fig. 4, A and B) in that they enhance the rate of proteolysis of the "A" region while they decrease the rate of trypsin digestion of the "B" region.
Optimum Protection of "B" Region by Poly(dT) against Tryptic Hydrolysis Occurs at a Basel32P Ratio near 5--The results in Table III suggest that optimal protection of the "B" region with single stranded fd DNA occurs at a ratio of 4 to 5 nucleotide bases per 32P monomer. Increasing the ratio of fd DNA/32P resulted in decreased protection. As shown in Fig.  5, the same phenomenon was also observed with poly(dT). Protection of the "B" region of 32P against trypsin digestion reached a maximum of 72% at a poly(dTY32P base ratio of 4.8. Decreased protection of the "B" region was observed if the base/32P ratio was increased above 4.8. The ratio of 4.8 bases/ 32P is very close to the estimated binding site size of five bases per protein monomer published by Kelly et al. (26). Various other estimates of the binding site size are 6.7 to 7.5, 10, and 11 bases per gene 32 molecule as reported by Jensen et al. (161,Alberts and Frey (3), and Anderson and Coleman (251,respectively. Aside from the variety of methods used to estimate the size of the binding site there is no apparent explanation for the wide variation reported. Short Oligonucleotides Do Not Affect Rate of Tryptic Hydrolysis of Gene 32 Protein -If the "B" region of 32P is directly involved in DNA binding then oligonucleotides too short to permit cooperative protein binding would still be expected to decrease the rate of tryptic cleavage of the NH, terminus. The fluorescence studies of Kelly et al. (26) suggest that oligonucle- The SDS-polyacrylamide gels were scanned with a Joyce-Loebl densitometer and the per cent of "A" or "ES" region released during the course of the digestion was determined as in Table IV. "A" region release was measured at a low trypsin concentration (0.04 pglml) and "B" region release was determined at a high trypsin concentration (8.0 Kg/ml).
Oligo-ortt)doelynucleo-Base concentra-"A" Region re-"B" Regmn retmn l eased with low l eased with high trv!mn trvrksn in each case were adjusted so that 95% of the 32P present was bound to the respective oligonucleotide (based on the binding constants4 reported by Kelly et al. (26)). The results shown in the last column of Table V reveal that short dT-containing oligonucleotides do not protect the "B" region from tryptic hydrolysis. In this study all of the 32P was converted to 32P*-(A + B) regardless of whether the digestion was done in the presence or absence of d(pT),, d(pT&, or d(pT),. The same study was repeated at a lower trypsin concentration to observe the effect of the same oligo(dT)containing nucleotides on the removal of the "A" region of 32P. The results tabulated in the third column in Table V reveal that short oligonucleotides do not enhance the rate of tryptic cleavage of the "A" region. DISCUSSION Our results show that cooperative binding of the gene 32 protein to DNA and to some homopolynucleotides increases the rate of tryptic cleavage of the "A" region and decreases the rate of trypsin hydrolysis of the "B" region. Neither of these effects are observed with short oligonucleotides (<8 bases) which still bind 32P. We have interpreted these results in terms of a model for the mode of 32P:DNA interaction (Fig. 6). The essential feature of the model requires that the gene 32 protein exists in at least two different conformations, only one of which is capable of cooperative binding to DNA. According to the work of Carroll et al. (6), gene 32 protein exists mainly as a dimer or higher aggregate forms at concentrations above 0.1 mg/ml even in the absence of DNA. Because of the results reported by Kelly and von Hippel (28) we have depicted the DNA binding sites as being partially occluded in the dime+ 4 The binding constant for d(pT), was assumed to be 1.5 x lo5 which is the constant reported for d(TpT) (26). This assumption is probably valid since d(ApA) and d(pA), both have identical association constants (26). The reported constants (26) for d(pT), and d(pT), were averaged to get an approximate constant for d(pT),. d(pT),,-,,, poly(dT), and fd DNA were presumed to have binding constants of at least 10" (26). 5 Kelly and von Hippel (28) 5). At the trypsin concentration used in these experiments the "A" region is removed from 32P within the first few minutes of the digestion. Hence, we are actually observing the effect of excess DNA or poly(dT) on the proteolytic removal of the "B" region from 32P*-A. Increasing the base/32P ratio above 5 actually results in decreased protection so that at a base/32P ratio of 15.4 there is only an 8% difference in the rate of proteolysis of the "B" region in the presence and absence of poly(dT) (Fig. 5). Since the gene 32 protein binds cooperatively to DNA (3, 5, 12), then even at a base/32P ratio of 15.4 the protein should still be bound in long clusters along the DNA.
We suggest that the decreased "B" region protection at base/32P ratios above 5 results from exchange of 32P*-A molecules from these long clusters to exposed sites on the poly(dT) added in excess of the amount required for stoichiometric binding of 32P. If the "B" region is not involved in 32P:32P cooperative interactions it is susceptible to digestion. The exchange of 32P*-A from a contiguous to a noncontiguous site on poly(dT) appears to occur rather slowly.
With a trypsin concentration of 16 pg/ml and the conditions used in Fig. 5, 90% of the 32P*-A is converted to 32P*-(A + B) in 10 min (data not shown).
In contrast, if poly(dT) is present in a base/32P ratio of 15.4 only 55% of the 32P*-A is converted to 32P*-(A + B) in this time. At 45 min, however, 88% of the "B" region is removed.
Nitrocellulose binding studies are in progress to compare 32P and 32P*-A with respect to their ability to bind DNA cooperatively and to exchange from a contiguous to a noncontiguous binding site on DNA. Moise and Hosoda (14) showed and we have confirmed that the "B" region is necessary for tight binding of 32P to singlestranded DNA. Our assignment of the "B" region at the NH, terminus is also consistent with the genetic studies of Breschkin and Mosig (32) and the in vitro studies of Huberman et al. (33). The latter group have found that the P7 mutant has a temperature-sensitive gene 32 protein which is defective in fl In contrast to the study by Bobst and Pan (291, Kelly et al. (26) reported the binding of 32P to oligo-and polynucleotides to be nonspecific with respect to base composition. However, Kelly et al. (26) did comment that the apparent discrepancy between the study of Bobst and Pan (29) and their own could be accounted for by binding constant and cooperativity differences for different polynucleotides well within the standard error of their measurements.