Developmental systems are controlled by modulating gene expression in response to internally programmed signals as well as to external signals. Our laboratory was interested in studying the molecular interactions and the signaling that occur to regulate gene expression. We exploited the genetic systems available in Escherichia coli (E. coli), its plasmids, and its viruses (e.g., bacteriophage lambda) to help us understand (1) gene regulation at the levels of transcription initiation and elongation, and translation initiation, and (2) cell growth and cell cycle control signals.

RECOMBINEERING:  We developed an in vivo cloning and gene modification system using lambda Red-mediated homologous recombination with short (<50 bp) homologies. This technology, called recombineering, was used as a great tool in E. coli for genetic modification. Over the years, we provided recombineering strains to more than 3,000 labs. Well over half of these labs worked on eukaryotic model systems like mouse and Drosophila. Modifications were made to clones in E. coli, which were then used to generate transgenics or targeted gene modifications in these model systems.

Recombineering eliminated the need to cut DNA with restriction enzymes or join DNA fragments with DNA ligase. This extremely efficient recombination system revolutionized the way in which recombinant DNA work was done and became a major tool in the field of synthetic biology. Visit our website on recombineering:  https://redrecombineering.ncifcrf.gov/

The N gene of lambda is the first gene expressed following viral infection. The function of the N protein is necessary for expression of most other lambda genes by its actions as a positive regulator. Positive activation of other genes occurs by N binding to specific RNA sites called NUT, modifying the RNA polymerase transcription complex. This modified polymerase complex reads through transcription terminators to distal lambda genes. Thus, the expression and action of N are central to the control of lambda development.

We determined that N is subject to novel posttranscriptional regulatory circuits. Expression of the N gene is autoregulated by N binding to the NUT RNA site 150 bases upstream of the N gene, thereby repressing the translation of N more than 100-fold over this long distance. The N-modified RNA polymerase complex is required for translational repression. Thus, antitermination and translation repression by N are coupled. This may be caused by a specific folding of the RNA structure that brings the NUT RNA into close juxtaposition with the N ribosome-binding site. RNaseIII, an endonuclease, recognizes the stem structure and cleaves it, separating NUT from the N RNA. This cleavage prevents N translational repression but actually enhances antitermination, presumably by releasing the antitermination complex from its interaction with the NUT RNA.

Additionally, we found that RNaseIII is expressed from an operon in which an essential low-molecular-weight GTP-binding protein, Era, is also encoded. From this operon, RNaseIII and Era expression is coordinately regulated and increases in relation to growth rate. This growth rate regulation of RNaseIII and Era occurs at the posttranscriptional level, but the mechanism remains unknown. The accumulation of adequate levels of Era is essential for cytokinesis to be completed and cell growth to continue. We speculated that a threshold level of Era must accumulate before Era-GTPase is activated by a cellular signal to cause cell division and to allow cell growth to continue. We believed RNaseIII and Era were key components that couple regulation of growth and the cell cycle.

Our collaborators included David Friedman, University of Michigan; Max Gottesman, Columbia University; William Margolin, University of Texas-Houston Medical School; Joel Stavans, Weizmann Institute of Science; and Sankar Adhya, Stuart Austin, Xinhua Ji, Ding Jin, Mikhail Kashlev, and Jeffrey Strathern, CCR.