cII Protein Research Articles

A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

IHF stimulation of λ cII gene expression is inhibited by the E. coli NusA protein

The effects of E. coli proteins Integrative Host Factor (IHF) and NusA on the regulation of lambda cII gene expression are presented. As reported previously (Peacock et al. [1984] Proc. Natl. Acad. Sci. USA 81, 6009-6013), IHF stimulates the DNA-directed in vitro synthesis of cII protein or its first dipeptide, fMet-Val. Whereas NusA, by itself, has no effect on cII expression, the presence of NusA inhibits the IHF-mediated stimulation of cII synthesis.

A CII-responsive promoter within the Q gene of bacteriophage lambda

A site within the phage λ Q gene shares homology with the CII-activated promoters, p E and p I, and is oriented in the direction opposite to that of Q gene transcription. A DNA fragment containing this site can serve as a template for CII-activated transcription in vitro. To ask if this presumptive CII control site functions as a CII-activated promoter in vivo, a restriction fragment containing this promoter has been cloned on a plasmid so that synthesis of β-galactosidase will be under its control. When CII protein is supplied in trans from a compatible plasmid, this promoter, designated p aQ is activated to produce β-galactosidase. A promoter positioned within the Q gene which can be activated by CII protein to initiate transcription in an anti-sense direction should result in an interference with Q gene expression, enhancing CII regulation of late functions, and adding to the list of known CII controls on the lysogenic response.

高トリグリセライド血症における超低比重リポ蛋白のアポ蛋白異常

The percentages of apo C-II and C-III proteins in very low density lipoprotein (VLDL) were high in the patients with hypertriglyceridemia (type IIb and IV) when compared with normal control subjects and those of apo A-I, A-II and B proteins were low. Positive correlation was shown between serum VLDL concentration and the percentage of apo C proteins, and the inverse relationship was between VLDL concentration and the percent apo B protein. Consequently, C/B and C/E ratios were higher in these patients than in healthy control.By contrast, there were no significant difference in high density lipoprotein apoprotein composition between normal subjects and the patients with high triglyceride concentration.

CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene

Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene ( galK), we have constructed a plasmid in which the λp re promoter controls galactokinase expression. A galK − host containing this plasmid has a Gal − phenotype since transcription from P RE requires activation by the A CII protein. When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein. A second plasmid was constructed in which the P RE promoter from phage21 controls galactokinase expression. Transcription of the galK gene in this plasmid requires the phage 21 CII protein. Using this system, we demonstrate that the λ and 21 P RE promoters are highly selective for their corresponding CII proteins. However, a cross-reaction between 21 P RE and the λ CII protein was observed. In addition, we transferred the P RE - galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy. Galactokinase expression in this single copy P RE - galK system is also dependent on CII protein, which may be provided from a multicopy plasmid. The high concentration of CII protein provided by the plasmid results in maximal expression of the P RE - galK transcription unit. In this second system low levels of CII activity from CII − mutants are amplified and can be readily detected.

Mutations that alter the DNA binding site for the bacteriophage lambda cII protein and affect the translation efficiency of the cII gene

The efficiency of translation of the cII gene of bacteriophage lambda is greatly reduced by the cII3059 mutation, a GUU → GAU (Val → Asp) change in the second cII codon. Mutations in the third and fourth codons of the cII gene, called ctr mutations, reverse this translation deficiency. Lambda cII3059 ctr-1, which has a GCA å ACA (Ala å Thr) change in the fourth cII codon, produces about half the normal level of cII activity in liquid cultures, and λcII3059 ctr-2 and λcII3059 ctr-3, which have identical CGT å CGC changes in the third codon, produce normal levels of cII activity in liquid culture. Since the cII protein of λcII3059 ctr-3 has the same primary sequence as that of λcII3059, the cII − phenotype of λcII3059 can be explained entirely by the deficiency of translating cII mRNA. We propose that ctr mutations increase translation efficiency by destabilizing a stable stem structure which can be formed by cII mRNA. The ctr mutations lie in an overlapping regulatory region which contains, in addition to sequence elements that influence the rate of cII translation, a region to which cII protein binds to activate transcription from the P RE promoter. The ctr-1 mutation alters the cII recognition sequence from 5′-T-T-G-C-N 6T-T-G- C -3′ to 5′-T-T-G-C-N 6T-T-G- T -3′, but has no effect on P RE activity. Since a C å T change in the first (5′-proximal) T-T-G-C sequence (to yield 5′-T-T-G- T -N 6T-T-G-C) greatly lowers cII binding affinity, cII protein must not recognize the two T-T-G-C sequences in an identical manner.

Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda.

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

Apoprotein C effect on triglyceride transport in tandem-perfused rat hind end and liver.

The metabolism of double-labeled triglyceride in a synthetic emulsion was defined in an in vitro perfusion system of rat hind end and liver described previously [Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G106-G112, 1983]. The metabolism of [3H]glycerol-[14C]triolein was defined in the absence of added apoproteins and with additions of human CII and both CII and CIII. Without apoprotein, a pronounced lipolysis of the triglyceride was recognized by high concentrations of radiolabeled glycerol and free fatty acid in the perfusate. The removal of an aliquot of hind-end venous effluent 5 min after adding the labeled triglyceride emulsion to the arterial inflow demonstrated a brisk lipolysis of the substrate when incubated outside the perfusion system. The addition of CII protein to the emulsion before its introduction into the tandem system eliminated perfusate lipolysis, both within the perfusion system and in incubations of aliquots withdrawn from the system. Intravascular lipolysis was not seen with triglyceride emulsions containing both CII and CIH or when an aliquot of hind-end venous effluent was incubated with triglycerides that had not been exposed to the perfusion system. The intravascular lipolysis observed for the [14C]triglyceride added to the tandem system without apoproteins was associated with relatively greater recoveries of 14C-fatty acyl in liver, fat, and muscle and relatively greater recoveries of 14CO2 than when CII alone or both CII and CIII were added with the triglyceride. The addition of CIII to CII in a 1:1 molar ratio increased the recovery of 14C-fatty acyl in muscle and the recovery as 14CO2.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of bacteriophage lambda CII activity by bacteriophage and host functions.

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.

Removal of a terminator structure by RNA processing regulates int gene expression

The int gene of phage λ encodes a protein involved in site-specific recombination. Its expression is regulated differentially during successive phases of the λ infective cycle. The gene is transcribed early after infection from one promoter, p L, and later from a second promoter p I. Each transcription event requires different positive activation factors, λ N and cII proteins, respectively. Transcription from the p I promoter, located adjacent to int, passes through int and terminates 277 nucleotides beyond int at t I. Polymerases initiating at p L transcribe through t I, and into the b segment of λ DNA. The read-through p L transcript is sensitive to cleavage by the endonuclease, RNase III, both in vivo and in vitro. Two specific cuts are made by RNase III in a double-stranded structure about 260 nucleotides beyond int in the location of the t I terminator. Functionally, the processed p L transcript is unable to synthesize the int gene product, whereas the terminated and unprocessed p I transcript expresses int. Interestingly, unprocessed p L transcripts made in hosts defective in RNase III ( rnc −) can express int. Thus a correlation exists between processing and negative control of int expression. The place where processing occurs, some 260 nucleotides beyond int, is called sib, and the control of int expression from this site is called retroregulation. Retroregulation by sib is not restricted just to the int gene; we show that if the sib site is cloned beyond a bacterial gene, the gene is controlled by sib and RNase III. Specific models are discussed with respect to control of gene expression by RNase III from a site beyond the controlled gene.

Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli.

High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.

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.

Mutational analysis of a regulatory region in bacteriophage lambda that has overlapping signals for the initiation of transcription and translation.

The positively regulated PRE promoter of phage lambda structurally overlaps with the ribosome-binding and NH2-terminal coding region of the regulatory protein (cII) that activates PRE transcription. We have isolated and characterized 27 different point mutations that occur within the 36-base-pair overlapping region. A comparison of genetic crossover data with nucleotide separations as determined by DNA sequence analysis reveals that recombination frequencies are greatly depressed at very short distances. Moreover, recombination frequency is critically dependent upon the precise nucleotide sequence of the crossover region for distances of five nucleotides or less. The mutations define precise positions and sequences that are important to (i) PRE promoter function, (ii) translation of the cII gene, and (iii) cII gene function. Mutational changes that affect the function of one element in this region concomitantly define phenotypically silent alterations in the other two elements. Mutations deficient in promoter function (P-RE or cy) are clustered in two regions that lie approximately equal to 10 and approximately equal to 35 nucleotides before the initial base of PRE mRNA, analogous to mutations in other promoters. P-RE mutations in the -10 region alter bases that are conserved in prokaryotic promoters, but P-RE mutations in the -35 region do not affect bases that are normally conserved in other promoters. Several mutations deficient in cII gene activity affect the initiation of cII protein synthesis, including an A leads to G change four bases outside the cII coding region, and AUG leads to GUG, AUG leads to ACG, and AUG leads to AUA mutations in the initiation codon. In the region of overlap between the PRE promoter and the NH2-terminal region of the cII gene, most amino acid substitutions in the cII protein do not result in a loss of cII function, indicating that this region of the gene does not contain essential information for cII function. We suggest that the overlap itself is an evolutionarily conserved structure and that it somehow coordinates the bidirectional transcriptional and translational events that occur in this region.

The SOS system of Escherichia coli in the regulation of bacteriophage λ development

A sequence homologous to the known SOS boxes is found in the P o promoter of phage λ. It is suggested that the sequence found is a binding site for the LexA repressor. The mechanism of the LexA part in regulation of λ development is discussed. It is based on the competitive transcription of the RNA encoding CII protein and the short OOP-RNA transcribed from the P o promoter.

Bacteriophage λ protein cII binds promoters on the opposite face of the DNA helix from RNA polymerase

The bacteriophage lambda transcriptional activator protein, cII, coordinately regulates transcription from two phage promoters that control lysogenic development. We demonstrate that cII is a DNA binding protein that selectively interacts with a repeat sequence in the -35 region of the promoter. Furthermore, cII is shown to bind mainly one face of the DNA helix and to make its contacts primarily in the major groove of the DNA. RNA polymerase sees this same region from the opposite side and sandwiches the DNA helix between itself and cII.

The integrase promoter and T′ I terminator in bacteriophasr λ and 434

The lambda integrase promoter PI lies immediately downstream from a terminator T'I. The PI promoter is activated by cII protein during lysogenization. The function of T'I is unknown. A deletion (trp-lambda 29), whose fusion point lies within one stem of T'1 retains cII-activable promoter function but shows little if any transcription termination in vivo. The nucleotide sequence of PI is identical in lambda and the related phage 434. However, the T'I sequences of the two phages, though located in the same position, are not detectably homologous. When restriction fragments carrying the promoter-terminator segments from each of these phages were inserted into the trp operon, both responded identically to cII activation, and both caused termination.

Probing cII and himA action at the integrase promoter p1 of bacteriophage lambda

Plasmids were constructed to supply cII-coded protein for activation of the phage promoter p 1. Using a fusion which expresses lacZ from p 1, we can accurately follow activation of p 1 without having to assay int activity in vivo. A large excess of cII protein compared to a normal lytic infection stimulates lacZ expression about 10-fold over the basal level. The int-c226 constitutive allele of p 1 is not further activated by cII even though its level of lacZ expression is less than the maximal cII-activated expression from wild type p 1. A himA-deleted strain also shows activation, demonstrating that there is no absolute requirement for the himA gene product for ell-stimulated transcription at p I .

Nucleotide sequence of the immunity region of bacteriophage Mu.

The leftmost 1590 bp of Mu DNA covering the immunity region have been sequenced. This region encodes the cI repressor, the cII or ner function and the beginning of gene A. An open reading frame extends from position 863 to 342 on the l-strand corresponding to cI protein with a molecular weight of 19212. It is preceded by a sequence resembling a promoter. To the right of the HindIII site an open reading frame extends from position 1099 to 1323 corresponding to cII or ner protein (molecular weight of 8505) followed by the beginning of gene A at position 1328. Between position 863 and 1099 promoters for leftward and rightward transcription and operator-like structures can be recognized in the sequence. The promoter for rightward transcription overlaps with the HindIII site and coincides with a RNA polymerase binding site as demonstrated by electron microscopy.

Proposed α-helical super-secondary structure associated with protein-DNA recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.

Purification and properties of a transcriptional activator. The cII protein of phage lambda.

We have purified the phage lambda transcriptional activator protein cII. The procedure described allows cII to be obtained in both high purity and yield, and thus allows detailed physical and chemical analysis. We demonstrate that cII in solution is a tetrameric protein and that it undergoes specific processing at its NH2-terminal end. In addition, the protein is characterized as to its molar extinction coefficient, molecular weight, amino acid composition, isoelectric point, alpha-helical content, and antigenic capability.