Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2020)

The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed, it is critical to the control of gene expression in bacteria. Despite the recognition of its importance to the cell’s physiology and metabolism, a detailed and comprehensive understanding of its mechanistic underpinning has remained elusive. In my thesis, I aimed to uncover potential routes of crosstalk between RNA polymerase and ribosome, the key players of transcription and translation, respectively. In my in vitro work, I identified direct interactions between both of these nanomachines as potential element for the regulation of the coupling of both processes. Using several methods I was able demonstrate the existence and specificity of this interaction and in collaboration with my colleagues from the Blaha and Wang laboratories, I was able to narrow the RNA polymerase binding interface on the ribosome to the head region of the small ribosomal subunit. All this progress in our understanding of transcription-translation coupling depended on a reliable method of separating free from ribosome-bound RNA polymerase. To this end I have established a reliable analytical procedure using sucrose gradient centrifugation followed by SDS polyacrylamide gel electrophoresis. Recently, I improved this procedure by replacing the sucrose gradient centrifugation with native gel electrophoresis, thus reducing the time and the amount of material required for the analysis of the interactions between RNA polymerase and ribosomes. To place my finding of the direct interaction between RNA polymerase and ribosomes in context of the resurgence of interest in the mechanism of coupling, I have included in this thesis the review I co-authored that summarizes the recent literature of the field. In this review, we integrate the presented data of the literature and of my work into a dynamic model of transcription-translation coupling in which the interactions between RNA polymerase and ribosomes are repeatedly formed and broken. The careful comparison of the effects of the direct interactions between RNA polymerase and ribosomes with those mediated by transcription factors NusG and RfaH leads us to propose two distinct modes of coupling: a factor-free and a factor-mediated coupling.

    Cover page: Direct Interactions Between Bacterial Ribosomes and RNA Polymerase
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2019)
    The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed. The discovery of physical interactions between RNA polymerase and ribosomes has spurred renewed interest into this long-standing paradigm of bacterial molecular biology. Here, we provide a concise presentation of recent insights gained from super-resolution microscopy, biochemical, and structural work, including cryo-EM studies. Based on the presented data, we put forward a dynamic model for the interaction between RNA polymerase and ribosomes, in which the interactions are repeatedly formed and broken. Furthermore, we propose that long intervening nascent RNA will loop out and away during the forming the interactions between the RNA polymerase and ribosomes. By comparing the effect of the direct interactions between RNA polymerase and ribosomes with those that transcription factors NusG and RfaH mediate, we submit that two distinct modes of coupling exist: Factor-free and factor-mediated coupling. Finally, we provide a possible framework for transcription-translation coupling and elude to some open questions in the field.
      Cover page: Two Old Dogs, One New Trick: A Review of RNA Polymerase and Ribosome Interactions during Transcription-Translation Coupling
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2021)
      Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.
        Cover page: Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2017)
        In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.
          Cover page: Transcription–translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2020)
          The Major Facilitator Superfamily (MFS) is currently the largest characterized superfamily of transmembrane secondary transport proteins. Its diverse members are found in essentially all organisms in the biosphere and function by uniport, symport, and/or antiport mechanisms. In 1993 we first named and described the MFS which then consisted of 5 previously known families that had not been known to be related, and by 2012 we had identified a total of 74 families, classified phylogenetically within the MFS, all of which included only transport proteins. This superfamily has since expanded to 89 families, all included under TC# 2.A.1, and a few transporter families outside of TC# 2.A.1 were identified as members of the MFS. In this study, we assign nine previously unclassified protein families in the Transporter Classification Database (TCDB; http://www.tcdb.org) to the MFS based on multiple criteria and bioinformatic methodologies. In addition, we find integral membrane domains distantly related to partial or full-length MFS permeases in Lysyl tRNA Synthases (TC# 9.B.111), Lysylphosphatidyl Glycerol Synthases (TC# 4.H.1), and cytochrome b561 transmembrane electron carriers (TC# 5.B.2). Sequence alignments, overlap of hydropathy plots, compatibility of repeat units, similarity of complexity profiles of transmembrane segments, shared protein domains and 3D structural similarities between transport proteins were analyzed to assist in inferring homology. The MFS now includes 105 families.
            Cover page: Expansion of the Major Facilitator Superfamily (MFS) to include novel transporters as well as transmembrane-acting enzymes
            Top