Gary N. Gussin

Genetic characterization of a prm− mutant of bacteriophage λ

Abstract It has been hypothesized ( Reichardt and Kaiser, 1971 ) that transcription of the λ genes c I and rex , which code for repressor and rex protein, respectively, can be initiated at either of two sites depending on whether or not repressor itself is present. On infection of a sensitive cell, transcription of repressor ( c I) and rex mRNA is thought to be initiated at a “promoter for establishment of repressor synthesis” ( pre ); however, in an established lysogen, in the presence of active repressor, synthesis of c I- rex mRNA appears to be initiated at a “promoter for maintenance of repressor synthesis” ( prm ), whose existence has been inferred from several indirect observations ( Bode and Kaiser, 1965 ; Reichardt and Kaiser, 1971 ; Echols and Green, 1971 ; Castelazzi et al. , 1972 ). In this communication, we describe the isolation of a mutant defective in prm , the promoter for synthesis of c I- rex mRNA in the presence of repressor (as in an established lysogen). The mutant, prm 116, maps at a single site between the c I gene and 0 R (the operator to which repressor binds to block rightward transcription of the λ x operon), is phenotypically c I − rex − , and yet complements the pre − mutant c Y42 for lysogenization. Thus, its existence precisely locates prm and provides a direct verification of some elements of the two-promoter hypothesis for c I- rex transcription. Since prm 116 does not appear to affect repressor binding at 0 R , the two sites, prm and 0 R , can be separated functionally by mutation.

Role of cII protein in stimulating transcription initiation at the λ PRE promoter: Enhanced formation and stabilization of open complexes

Abortive and productive initiation assays were used to study transcription initiation at the PRE promoter of phage lambda in vitro. Two parameters were measured: k2, the rate constant for the transition between closed and open complexes; and KB, the equilibrium constant for the initial binding of RNA polymerase to promoter DNA. In the absence of cII protein (which activates PRE) the PRE promoter was extremely weak as expected, with k2 = 4.0 X 10(-4) S-1 and KB = 1.0 X 10(7) M-1. The addition of cII protein resulted in about a 15-fold increase in KB and a 40-fold increase in k2. Thus, cII activation of PRE results both in enhanced binding of RNA polymerase to DNA to form closed complexes and in an enchanced rate of isomerization of closed to open complexes. In addition, we found that open complexes formed in the presence of cII protein were at least four times as stable as those formed in its absence. This suggests that RNA polymerase and cII protein may remain in close contact even after complexes are formed.

Differential effects of mutations on discrete steps in transcription initiation at the λ PRE promoter

Abstract The effects of cy mutations on transcription initiation at the λ P RE promoter were determined using abortive initiation analysis (McClure, 1980). In the presence of λ cll protein, which activates P RE , three mutations in the −10 region dramatically reduce k 2 , the forward rate constant for the isomerization of closed to open complexes, but only slightly affect K B , the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes. In contrast, five −35 region mutations caused decreases of 30 to 150 times in K B with much smaller effects on K 2 . In the absence of cll protein, the effects of mutations in the −10 region are qualitatively similar to those observed in the presence of cll protein, although the reductions in K 2 are much less dramatic. In contrast, none of the mutants with defects in the −35 region is distinguishable from wildtype P RE in the absence of cll protein. Thus RNA polymerase may recognize different sequences in the −35 region in the absence of cll protein than in its presence.

The role of gene cro in phage development

Abstract Phage λ cro mutants are unable to grow at 42°. This is due to two effects. One effect, called Tro , prevents the growth of other heteroimmune lamboid phages and is expressed by phage i λ c I857 cro 27 bio 252 but not by i λ c I857 cro 27 bio 256. The other effect interferes with phage growth only in cis and is expressed by phage i λ c I857 cro 27 bio 256.

Nucleotide sequences of the trpI, trpB, and trpA genes of Pseudomonas syringae: positive control unique to fluorescent pseudomonads

A 904-bp probe from Pseudomonas aeruginosa was used to identify the trpB, trpA and trpI genes of Pseudomonas syringae. Transcription initiation at the P. syringae trpBA promoter in vitro was activated by the P. aeruginosa TrpI protein in the presence of indoleglycerol phosphate. Thus, trpB and trpA are regulated positively in three species of fluorescent pseudomonads, P. aeruginosa, P. putida, and P. syringae, but in no other eubacteria so far investigated [Crawford, Annu. Rev. Microbiol. 43 (1989) 567-600]. In addition to conservation of protein-coding sequences, there is a high degree of nucleotide sequence identity in the intergenic control region that includes the divergent trpI and trpBA promoters, especially in the binding sites for TrpI protein. Differences in patterns of codon usage distinguish the trpI genes of P. syringae and P. putida from P. aeruginosa trpI and from the trpB and trpA genes of all three species.

DNA sequence analysis of prm- mutations of coliphage lambda

Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda have been determined. Prm-mutations have been assigned to two classes. Class I mutations appear to affect the interaction of RNA polymerase with prm; six class I mutations affect four sites, located 14, 33, 38, and 39 bp preceding the prm transcription startpoint. Class II mutations appear to owe their Prm-phenotype to a change in OR, which could prevent activation of prm by repressor. All three class II mutations are in OR 1.

Characterization of a doubly mutant derivative of the λ PRM promoter: effects of mutations on activation of PRM

The mutation, prmE37, located at —14 in the PRM promoter of bacteriophage λ, reduces PRM function dramatically both in vitro and in vivo. In a search for second-site revertants of prmE37, we isolated a double mutant that exhibits a partially restored Prm+ phenotype. The second-site mutation (at —31) is identical to the mutation prmup-1. The activity of the doubly mutant (pseudo-revertant) promoter, prmE37prmup-1, was investigated in vivo using a PRM-lacZ fusion phage and found to be intermediate between that of prmE37 and wild-type PRM. However, the relative strength of the prmE37prmup-1 promoter was greater than expected following superinfection of a λ lysogen. Since nalidixic acid was found to preferentially inhibit transcription from the doubly mutant promoter under these conditions, we suggest that DNA supercoiling favors activation of this promoter by represser. In runoff transcription assays in the absence of repressor, the activity of wild-type PRM and the doubly mutant promoter were the same. However, while addition of repressor significantly stimulated wild-type PRM, it had little or no effect on the activity of the doubly mutant promoter. Values of KB, the equilibrium constant for formation of closed complexes, and kf, the rate constant for isomerization of closed to open complexes, were determined in abortive initiation assays, and the product kfKB was used as a measure of promoter strength. The results of these assays are in agreement with those obtained in runoff transcription assays. In the absence of repressor, values of kfKB for the doubly mutant promoter and wild-type PRM are the same; however, τobs, the time required for open complex formation, is significantly greater for the double mutant than for wild-type PRM at all RNA polymerase concentrations used for the abortive initiation analysis. In the presence of repressor, the doubly mutant promoter is stronger than the prmE37 promoter, but much weaker than wild-type PRM. This is due to the fact that kf for the doubly mutant promoter is increased 2.5-fold by repressor, but KB is reduced to the same extent. These two effects counteract each other, so that repressor has no net effect on the strength of the prmE37prmup-1 promoter in vitro. In contrast, repressor increases kf for wild-type PRM eightfold and increases overall promoter strength (KBKf) nearly fivefold. In the presence of repressor, the effects of the two mutations, prm E37 and prm up-1, on kf are independent. This observation is discussed in relation to revised models for open complex formation.

In vitro transcription of adenovirus 2 DNA

SummaryE. coli RNA polymerase holoenzyme is able to recognize transcription initiation sites on Adenovirus 2 DNA that are functionally indistinguishable from promoters for the enzyme on phage DNAs. The complexes formed between the polymerase and the DNA at these sites can exist in two states — either as I (initiation) complexes, from which rapid RNA chain initiation is not possible, or as RS (rapid starting) “rifampicin resistant” complexes, from which rapid RNA chain initiation can occur. When transcription is limited to that initiated from stable, rifampicin-resistant pre-initiation complexes, initiation is strictly dependent on the presence of sigma factor; in addition, the frequency of initiation exhibits sigmoidal dependence on the temperature at which pre-initiation complexes are allowed to form, with a transition temperature of 26–28°C. The average half-time for initiation of RNA chains from sites on Ad 2 DNA is shown to be comparable to half-times for initiation of RNA chains from promoters on T7 and λ DNAs. At saturating levels of enzyme, the half-times are 0.6, 0.9, and 1.6 sec for λ b2, Ad 2 and T7 DNAs, respectively.The existence of efficient, phage-like promoters for E. coli RNA polymerase on Ad 2 DNA suggests to us that such promoters may be closely related functionally and spatially to promoters for mammalian RNA polymerases.

Location of Binding Sites for RNA Polymerase II from Wheat Germ and from Human Placenta on Adenovirus 2 DNA

SummaryElectron microscopic visualization of binary complexes between eukaryotic RNA polymerases and Adenovirus 2 (Ad 2) DNA was used to locate specific binding sites for the enzymes. RNA polymerase II from human placenta binds to 10–16 distinct sites depending on the ratio of enzyme to DNA and the divalent cation present in the binding mixture. Wheat germ RNA polymerase binds to 12–14 strong binding sites and 2–3 weaker sites, all but one of which correspond to binding sites for the placental enzyme. At least six of the strong binding sites for both enzymes correspond to promoters known to be active in vivo.As a test of the two-state model for transcription initiation, we examined binding of wheat germ RNA polymerase II to Ad 2 DNA at 0° and 37°. The extent of binding was the same at the two temperatures and the distributions of binary complexes were virtually identical. This observation, in conjunction with results presented previously, is strong support for the existence of I and RS complexes in eukaryotic systems.

Clustering of Prm− mutations of bacteriophage λ in the region between 33 and 40 nucleotides from the cl transcription start point

Abstract Mutations affecting P RM -directed expression of the λ c I- rex operon have been isolated and characterized. Six (class I) mutations, including prm 116 ( K.-M. Yen and G. N. Gussin, 1973 , Virology 56 , 300–312), appear to be defective in the interaction between RNA polymerase and the P RM promoter. Five of the six mutations map in the RNA polymerase “recognition” region ( W. Gilbert, 1976 , In “RNA Polymerase” (R. Losick and M. Chamberlin, eds.), pp. 193–205. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.) located approximately 35 nucleotides preceding the transcription start point, while one maps near the so-called Pribnow box, a heptanucleotide sequence common to some extent in all promoters sequenced thus far. The existence of mutations in two distinct regions of the promoter may provide the opportunity to distinguish between the functions of the two regions in transcription initiation. Three of the Prm − mutations (class II) are defective in repressor binding at O R . These mutations may be Prm − because repressor is unable to act as a positive regulator of P RM ( M. Ptashne, K. Backman, M. Z. Humayun, A. Jeffrey, R. Maurer, B. Meyer, and R. T. Sauer, 1976 , Science 199 , 156–161). A 10th mutant that exhibited some of the properties expected of a prm − mutant appears to be defective in the c I gene itself; most likely it is a c I missense mutant.