Autoregulation and Function of a Repressor in Bacteriophage Lambda

We present a statistical method for detection of palindromes in mRNA or DNA, starting from the protein sequence. Analysis of immunoglobulin genes by this method demonstrates that palindromic se uences are not randomly distributed. They are located at each side of the hypervariable regions in the variable (V) genes, whereas no such regular design is observed in the constant (C) genes. In addition, palindromic sequences overlap the V-C junction in all immunoglobulin classes and significant palindromes are present near residue 216 of the heavy chain, which is the end of deletions in many heavy chain diseases. The relevance of these palindromes to gene translocation and generation of diversity in antibodies is discussed.A palindromic DNA sequence possesses a 2-fold rotational axis of symmetry perpendicular to the-helix and, as a consequence, the base sequences of the two strands are identical when read in the same polarity:Such DNA sequences are universally distributed and have been shown to exist in bacteriophages (1, 2), in Escherichia coli plasmids (3), and in eukaryotic DNA (4). They can be rather long (300-1200 nucleotide pairs), and some are able to form hairpin-like structures in single-stranded DNA (4). They can also be found in heterogeneous nuclear RNA (5), which is characterized by its resistance to RNase. It has been suggested (6) that their structure (cruciform-like DNA) might be a recognition site for proteins acting at the DNA level; on this basis, molecular models for genetic recombination have also been proposed (7). These speculations are supported by several findings: the Lac operator is double stranded and about 27 base pairs long; and the sequence of its RNA transcription copy has been determined (8) and contains a partial 2-fold rotational symmetry. In phage X, the gene cl is surrounded by three left operators and three right operators. Each operator has an axis of partial 2-fold symmetry (9). Inverted repeats have been found in transposable DNA segments of bacteria (10,11 pointed out that palindromic sequences may occur near the V-C junction and that these sequences could be recognition sites for enzymes involved in the translocation process (14).In this paper, we present a method for detection of palindromes in mRNA, starting from the protein sequences. The method is applied to the case of immunoglobulins, and we show that the palindromic sequences are not randomly distributed but are located preferentially at the boundary of hypervariable regions and near the V-C junction. METHODS The searching procedure is an extension of Fitch's polypeptide comparison method (15): the protein sequence is translated into its degenerate mRNA sequence, which is then analyzed for the presence of palindromes. This analysis is performed by matching, in a sliding process, each subsequence of 11 nucleotides (polarity 5'-3') with all the possible consecutive subsequences of 11 nucleotides (polarity 3'-5'). For each match, a complementation index and its probability for occurring by chance are computed and ...

Section: ' G-g-g-a-a-a-t-t-t-c-c-c 3' 3' C-c-c-t-t-t-a-a-a-g-g-g 5'mentioning

confidence: 99%

On the location of palindromes in immunoglobulin genes.

Wuilmart¹,

Urbain²

1977

“…At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1,10) (Fig.…”

mentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...

“…Direct repeats function in regulating the expression of the cI and crc genes in phage A (30,31). The CI and Cro repressor proteins bind to the 17-bp repeats that constitute the left-and right-hand operators and promoters of the phage (32). It has been proposed that this binding serves to autoregulate transcription of these repressor genes (32).…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.

Stalker¹,

Kolter²,

Helinski³

1979

110

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K R6K is a multicopy (10-15 copies per chromosome) plasmid that is 38 kilobases (kb) in size and specifies resistance to the antibiotics ampicillin and streptomycin (1). A physical and genetic map of plasmid R6K has been obtained that includes two bidirectional origins of replication (2-4) and an asymmetric terminus of replication (2, 4). Construction of low molecular weight derivatives of R6K has defined a 2.1-kb segment of this plasmid that contains sufficient information for autonomous replication in Escherichia coli (see Fig. 1) (5). This segment contains an origin of replication and a region (pir) that codes for a protein (designated ir) that is required for initiation of R6K DNA replication (6-8). The pir region is included in two adjacent HindIII fragments (designated 15 and 9) in this 2.1-kb segment of R6K. The origin of replication is located in the region of the junction of the HindIII fragment 9 and the adjacent HindIII fragment 4. HindIII fragments 15 and 9 do not in themselves constitute a functional replicon, but when these two fragments are joined to a CoIEl replicon or to a X phage derivative integrated in the E. coli chromosome, they will support in trans the replication of origin-containing derivatives of R6K (8). These origin-containing derivatives have in common a 380-base-pair (bp) region of R6K that includes the junction of HindIII fragments 9 and 4 (Fig. 1). The nucleotide sequence of a portion of one of these origin-containing derivatives, pRK526, is described in this study. This nucleotide sequence includes a functional origin of replication of R6K and the region adjacent to the start of the structural gene for the 7r protein. MATERIALS AND METHODSEnzymes and DNA Preparations. The restriction endonucleases HindIII, Hae II, and Hae III were purified by the procedure of Greene et al. (9). Bgl II was the generous gift of C. Yanofsky. Bacterial alkaline phosphatase and T4 polynucleotide kinase were from Worthington and P-L Biochemicals, respectively. The Klenow fragment ("large fragment") of E. Plasmid pRK526 was maintained in the E. coli strain C600trpE5 (<80) (X*pirR6K) that was constructed by R. Kolter. The covalently-closed circular DNA form of pRK526 was isolated from Sarkosyl lysates (10) and purified by two centrifugations to equilibrium in CsCl/ethidium bromide gradients. The DNA was precipitated with isopropanol and dissolved in the appropriate buffers for restriction enzyme digestion and end-labeling.Labeling of DNA Fragments. Samples (10-20 jsg) of pRK526 were digested with either HindIII or Bgl II prior to end-labeling. The 5' end was labeled by means of 40 units of polynucleotide kinase (11, 12) and 1 ,uM [Ly-32P]ATP (3000 Ci/mmol, 1 Ci = 3.70 X 1010 Bq) in kinase buffer (50 mM glycine, pH 9.5/10 mM dithiothreitol/25% glycerol/0.2 mM spermidine) after dephosphorylation of the 5' end of the DNA with bacterial alkaline phosphatase. Incubations were for 30 min at 370C. Labeling the 3' ends by filling in 5'-over...