Recent advances in prokaryotic peptide transport

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25590/1/0000134.pd

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the oilier (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they arc the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38447/1/240290305_ftp.pd

Cloning, expression, and nucleotide sequence of livR , the repressor for high-affinity branched-chain amino acid transport in Escherichia coli

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggests that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38516/1/340010204_ftp.pd

Regulation Of High-affinity Branched Chain Amino Acid Transport In Escherichia Coli (repressor, Dna-binding Protein, Sequence, Leucine Regulation).

The genes for branched-chain amino-acid transport in Escherichia coli have been cloned into plasmids and the transport genes order, organization and DNA sequences have been determined. The expression of these genes is regulated by leucine primarily through a trans-acting negative controlling element. This dissertation presents the cloning of the regulatory gene and the identification of it by complementation analysis with strains of bacteria carrying mutations in transport regulation. The DNA sequence of the gene was determined and the calculated MW of the regulatory product is 19,155. The cloned regulatory gene has been transformed into E. coli minicells and the regulatory protein is strongly expressed in vivo. The secondary structure prediction for the regulatory protein indicates that the carboxy terminus may fold into several alpha helices. Two of these predicted alpha helices show sequence homology to DNA-binding proteins.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127787/2/8600400.pd