Biomedical subjects
Publications and source records attributed to R T Sauer.
One- and two-dimensional proton NMR methods are being used to study the synthetic lambda operator site O-L1, a 17 base-pair DNA duplex recognized by lambda repressor and Cro protein. The complete assignment of the 17 imino protons, which participate in Watson-Crick hydrogen bonding, and of the eight adenine H2 protons, which lie in the minor groove of the double helix, is presented.
The solution structure and dynamics of the 17-base-pair synthetic operator site OL1, which is recognized by the cI and Cro repressors of bacteriophage lambda, is studied by two-dimensional NMR methods. A sequential assignment strategy for nucleic acids is proposed and illustrated by the assignments of the base and sugar protons of OL1.
The thermal stabilities of mutant phage lambda repressors that have single amino acid replacements in the NH2-terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solvent-exposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.
Several general principles emerge from the studies of Cro, lambda repressor, and CAP. The DNA-binding sites are recognized in a form similar to B-DNA. They do not form cruciforms or other novel DNA structures. There seem to be proteins that bind left-handed Z-DNA (87) and DNA in other conformations, but it remains to be seen how these structures are recognized or how proteins recognize specific sequences in single-stranded DNA. Cro, repressor, and CAP use symmetrically related subunits to interact with two-fold related sites in the operator sequences. Many other DNA-binding proteins are dimers or tetramers and their operator sequences have approximate two-fold symmetry. It seems likely that these proteins will, like Cro, repressor, and CAP, form symmetric complexes. However, there is no requirement for symmetry in protein-DNA interactions. Some sequence-specific DNA-binding proteins, like RNA polymerase, do not have symmetrically related subunits and do not bind to symmetric recognition sequences. Cro, repressor, and CAP use alpha-helices for many of the contacts between side chains and bases in the major groove. An adjacent alpha-helical region contacts the DNA backbone and may help to orient the "recognition" helices. This use of alpha-helical regions for DNA binding appears to be a common mode of recognition. Most of the contacts made by Cro, repressor, and CAP occur on one side of the double helix. However, lambda repressor contacts both sides of the double helix by using a flexible region of protein to wrap around the DNA. Recognition of specific base sequences involves hydrogen bonds and van der Waals interactions between side chains and the edges of base pairs. These specific interactions, together with backbone interactions and electrostatic interactions, stabilize the protein-DNA complexes. The current models for the complexes of Cro, repressor, and CAP with operator DNA are probably fundamentally correct, but it should be emphasized that model building alone, even when coupled with genetic and biochemical studies, cannot be expected to provide a completely reliable "high-resolution" view of the protein-DNA complex. For example, the use of standard B-DNA geometry for the operator is clearly an approximation.(ABSTRACT TRUNCATED AT 400 WORDS)
To learn about the V lambda gene segments that are expressed in lambda 3 light chains, the most recently identified lambda-chain subtype in inbred mice, we determined partial amino acid sequences of the V regions of two of these chains, L5-8 and Lc49 . The partial sequences were extended by establishing the complete V region sequence of cDNA for the lambda-chain mRNA from the hybridoma (RZ 5-8) and the myeloma ( CBPC -49) that produce these chains. The primer extension method used to sequence the cDNA is described in detail, because the same primer (a synthetic heptadecadeoxynucleotide ) can be used for sequencing cDNA for lambda-chains of all three subtypes of inbred mice and probably for lambda-chains from some other vertebrate species as well. The results confirm earlier preliminary findings that for both chains the V region is encoded by the V lambda 1 and J lambda 3 gene segments. The unmutated germ-line sequences of these gene segments are present in both chains, but the two chains, nevertheless, differ at codon 97, the V lambda-J lambda boundary. A T/G difference in the third position of this codon resulted in a codon for histidine (CAT) in one chain (L5-8) and for glutamine (CAG) in the other chain ( Lc49 ). This difference can be accounted for by variation in the site of V lambda 1-J lambda 3 recombination. Though the V region amino acid sequences of L5-8 and Lc49 differ only by the His/Gln substitution at position 97, the two chains have been shown (manuscript in preparation) to differ in their ability to form an effective combining site for the 2,4-dinitrophenyl group.
The structure and activities of the recombination-promoting P22 Erf protein were examined in vitro. Treatment of the protein with elastase produces a stable amino-terminal fragment, consisting of amino acid residues 1 to (approximately) 136. We have purified this fragment, designated fragment B, to apparent homogeneity by gel filtration chromatography. Fragment B retains the oligomeric structure and single-stranded DNA binding specificity of intact Erf. It differs, however, in lacking the ability of intact Erf to bind single-stranded DNA into large aggregates following mild heat treatment of the protein. In addition, its binding to DNA may be weaker than that of intact Erf. Intact Erf sediments through a sucrose gradient as a discrete species with an apparent S20,w of approximately 11 X 7 S. Its sedimentation behavior is affected little, if at all, by concentration. Fragment B also sediments as a discrete species at approximately 10 X 4 S. In the electron microscope, intact Erf appears as rings, with 10 to 14 small projecting structures resembling the teeth of a gear. Fragment B is similar, except that it appears to lack the peripheral structures. From these observations, we conclude that Erf consists of at least two structurally and functionally distinct domains, and that it has a discrete ring-like oligomeric structure.
The c2 repressor of bacteriophage P22 can be digested with trypsin, chymotrypsin, or elastase to yield stable fragments. Purified NH2-terminal fragments, like intact repressor, bind specifically to P22 operator DNA and also mediate positive and negative control of transcription. COOH-terminal fragments of repressor do not bind operator DNA but do undergo a concentration-dependent oligomerization similar to that observed with intact repressor. These results suggest that P22 repressor, like the related cI repressor of phage lambda, contains two structural domains which mediate different functions of the intact molecule.
The DNA sequence of the immI immunity region of bacteriophage P22 has been determined. This region includes the ant gene, which encodes the P22 antirepressor protein, and the mnt and arc genes, which encode proteins that negatively regulate antirepressor synthesis. We have purified antirepressor protein and selected tryptic peptides of antirepressor, and have determined the amino terminal sequences and amino acid composition of these molecules. These data, in combination with the DNA sequence, locate the ant gene and define the complete amino acid sequence of antirepressor (300 residues). The mnt and arc genes have been located by sequencing the mnt-am343 and arc-amH1605 mutations. The Mnt and Arc proteins are predicted to be small, basic polypeptides that are homologous in amino acid sequence. The Mnt protein also shows significant sequence homology with the lambda Cro protein. The arc and ant genes are transcribed rightward from the Pant promoter, while mnt is transcribed leftward from a promotor that may overlap Pant. The Mnt protein apparently acts by binding to an operator site located immediately adjacent to the startpoint of Pant transcription.
The structural gene for Salmonella bacteriophage P22 tail protein, gene 9, is separated from the remainder of the P22 late operon genes by the immI region. Early transcription of immI gene ant, which is immediately promoter proximal to gene 9, occurs in the same direction as late gene transcription but does not enter gene 9 coding sequences (Susskind & Youderian, 1982). We have cloned gene 9 and surrounding sequences into pBR322 and subsequently positioned lac UV5 promoters at varying distances before the start of gene 9 by DNA manipulations in vitro. Using an in vitro phage assembly assay to measure in vivo expression of tail protein from these plasmids and in vitro transcription reactions to measure transcriptional template activity of DNA fragments isolated from these plasmids, we have identified a region of DNA between gene ant and gene 9 that behaves as a transcription termination signal. The DNA sequence of this region shows hyphenated dyad symmetry followed by a run of seven thymine residues on the coding strand. This sequence can be drawn in a potential stem-and-loop secondary structure similar to known rho-independent transcription termination signal sequences. We discuss the role of this transcriptional terminator sequence in gene 9 expression and the early to late transcriptional switch in the P22 infection cycle.
The Mnt repressor of Salmonella phage P22 recognizes a 17 base pair operator. We constructed an Mnt binding site with two symmetric operator-constitutive mutations. We then selected Mnt mutants that have lost the ability to bind the wild-type operator but acquired the ability to bind the mutant operator. Four independent mutations change the same CAC codon in the mnt gene to CCT or CCC. The corresponding amino acid change in Mnt is His6 leads to Pro. DNA binding assays with purified wild-type and mutant proteins confirm the change of binding specificity in vitro. Wild-type Mnt binds strongly to the wild-type operator, but binds the mutant operator with 1000-fold less affinity. The mutant Mnt binds with converse affinities to the two operators.
The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.
Using a combination of two and one-dimensional NMR spectroscopy, it is shown that in the intact bacteriophage lambda repressor, the N-terminal domain assumes the same global structure as when it remains isolated. It is further shown that the N-terminal domain is only loosely attached to the C-terminal domain in the intact repressor.
The lambda cI repressor and the P22 c2 repressor contain two structural domains. In both proteins, the N-terminal domains mediate operator recognition and positive control of transcription, and the C-terminal domains mediate subunit oligomerization and recognition of the recA protein. In some cases, structural, biochemical, and genetic studies implicate particular repressor side chains in these processes.
We have characterized about 50 different amino acid substitutions in the aminoterminal domain of lambda repressor. Sixteen of these substitutions alter external side chains of the repressor and cause a substantial reduction in the affinity of the mutant repressor for operator DNA. Seven of these mutant repressors were purified and were shown to be stably folded. The strong, external repressor mutations occur near the aminoterminal end of alpha helix 2, throughout alpha helix 3, and in the aminoterminal-arm region of the repressor. These results suggest that these regions of lambda repressor are close to operator DNA in the protein-DNA complex and thus that these regions comprise the DNA-binding sites of the repressor. Our genetic results support and are completely consistent with more-detailed models of the repressor-operator interaction based on model-building (Pabo and Lewis 1982; Lewis et al. this volume) and biochemical studies (Pabo et al. 1982).
Glycyl-tRNA synthetase is one of two Escherichia coli aminoacyl tRNA synthetases which has two different subunits. A 5.1-kilobase pair HindIII chromosomal DNA fragment was isolated, cloned into pBR322 (to give plasmid pTK201), and shown to direct synthesis in maxicells of both subunits (Mr = 35,000 (alpha) and Mr = 65,000 (beta) of glycyl-tRNA synthetase. Locations of alpha- and beta-subunit coding regions were established by introduction of Tn5 insertions into various positions within the 5.1-kilobase pair HindIII segment of pTK201 and by determining the effect of each Tn5 insertion on synthesis of alpha- and beta-subunits and on enzymatic activity. From the Tn5 insertion analysis, regions encoding the NH2 terminus of the alpha-subunit and of the beta-subunit were approximately defined and these regions were sequenced. To locate rigorously the respective NH2-terminal encoding sections in the DNA sequence, NH2-terminal amino acid sequences of alpha- and beta-subunits were established by standard Edman degradations and these sequences were aligned with the DNA sequence. This analysis established the following: 1) coding regions for the subunits are in tandem; 2) a single promoter is used for transcription of both coding sections and the order of transcription is from alpha to beta; 3) in the 500 nucleotides 5' to the start of the alpha-subunit coding section, there is no sequence arrangement like that found for regulatory regions of bacterial amino acid biosynthetic operons; 4) nine nucleotides serve as the spacer between the TAA stop of the alpha- and the ATG start of the beta-subunit coding regions, thus making both coding regions in the same reading frame; and 5) the TAA stop of the alpha-subunit and the next for nucleotides associated with the intersubunit region are complementary to the 3'-end of 16 S rRNA; this arrangement suggests ribosome re-initiation in the spacer region gives balanced synthesis of both subunits.
The tail structure of the Salmonella phage P22 mediates both adsorption of the phage to its host and enzymatic hydrolysis of the bacterial O-antigen. The tail is an oligomeric structure, which is assembled from a single polypeptide species. We report here the amino- and carboxyl-terminal sequences of the P22 tail protein and the nucleotide sequence of its gene (gene 9). These data specify the complete amino acid sequence of the tail protein. The tail protein is a slightly acidic protein containing 666 amino acids. Comparison of the gene and protein sequences indicates that mature tail protein arises by cleavage of the initiator N-formyl-methionine from the nascent chain.
From differences in the ability of fragments of the lambda repressor to protect operator bases from chemical modification we suggest that the first few N-terminal residues of the lambda repressor form an extended arm that reaches around the back of the DNA helix when repressor binds to the operator.
The amino acid sequences of the repressor and cro proteins of phages lambda, 434 and P22 are homologous, especially in a region in which repressor and lambda cro have a similar alpha-helix-turn-alpha-helix secondary structure. Model-building studies indicate that this structure is important in DNA binding, and we suggest it may be a common feature of many DNa-binding proteins.