Source: Dissertation Abstracts International, Volume: 75-05(E), Section: B.

Adviser: Thomas A. Steitz.

The bacterial ribosome is one of the most common targets of clinically used antibiotics. Structures of macrolide antibiotics in complex with the ribosome have shown that these molecules bind in the ribosomal exit tunnel and partially occlude the path of newly synthesized proteins as they move to the cytosol. However, discrepancies between various structural models of macrolide antibiotics in complex with the ribosome have prompted a revisiting of these structures in the context of the bacterial ribosome from T. thermophilus, a thermophilic bacterium closely related to E. coli. The macrolide-ribosome complex structures presented in Chapter 2 of this work show that macrolide antibiotics bind in a conserved manner consistent with structural work carried out with mutant ribosomes from the archaeon, H. marismortui, but inconsistent with structure-based models from the bacterium D. radiodurans. Additional structural work with the antibiotic chloramphenicol in complex with the T. thermophilus ribosome shows that the previously published model of chloramphenicol in complex with the D. radiodurans ribosome is likely to be incorrect.

Structures of two additional classes of antibiotics that act through novel mechanisms are presented: thermorubin and dityromycin. Thermorubin is a relatively understudied ribosome inhibitor first discovered in the 1970s, characterized in the 1980s and all but forgotten in the intervening decades. Seeking to understand its role as a translation inhibitor, we determined a 3.2 A resolution crystal structure of thermorubin in complex with the bacterial ribosome from T. thermophilus. Our structure, presented in Chapter 3, shows that thermorubin binds at the interface of the small and large ribosomal subunits near the binding sites of viomycin, paromomycin and hygromycin B. Despite its proximity to these other antibiotics, thermorubin appears to act through an entirely novel mechanism; it stimulates the rearrangement of several bases in the decoding center of the ribosomal A-site, sterically blocking the accommodation of a tRNA into the A-site. This structure and proposed mechanism is consistent with available biochemical data and allows us to outline a novel pathway through which antibiotics can be used to inhibit bacterial translation.

Dityromycin acts through an entirely different mechanism: it inhibits the EF-G catalyzed translocation of tRNA and mRNA through the ribosome. In Chapter 4 we present the structure of dityromycin in complex with the bacterial ribosome from T. thermophilus. Our structure reveals that dityromycin binds to protein S12 on the small ribosomal subunit, blocking a critical contact between EF-G, bound in the post-translocational conformation, and the ribosome. Our structure allows us to propose a mechanism for dityromycin inhibition whereby it traps EF-G in a pre-translocational conformation by blocking domain III of EF-G from adopting an orientation suitable for the placement of domain IV into the A-site, a critical step in EF-G catalyzed translocation. The mechanism of action of dityromycin proposed here is distinct from any known class of antibiotic, highlighting a novel site on the bacterial ribosome that can be exploited for the future development of novel antibiotics.

In Chapter 5, protocols used to obtain ribosome crystals capable of consistently diffracting to ∼3A resolution are discussed. In Chapter 6, we outline the development of an in vitro translation system which can be used to generate translating T. thermophilus ribosomes suitable for crystallographic studies. Because nascent peptide mediated translation arrest is an integral part of antibiotic resistance and its mechanism(s) at the structural level is not well understood, such a translation system is a first step towards determining the crystal structure of a ribosome-nascent chain complex.

Chapter 7 deals with the determination of the crystal structure of a domain of MALAT1, a highly conserved human noncoding RNA found to control the metastasis of human lung cancer. This lncRNA possesses a 3'-terminal region that has been shown to be important for stability, nuclear localization, and cancer proliferation. We present the crystal structure of this 3' terminal region and show how it is able to protect MALAT1 from degradation by nuclear exonucleases. Our findings demonstrate how a CGC/CG element within the 3' triple helix enables the precise placement of the 3' end MALAT1 to evade degradation by 3'-to-5' exonucleases. Our work also shows that the 3' region of MALAT1 adopts a structure similar to the Kaposi's sarcoma-associated herpesvirus expression and nuclear retention element (KSHV ENE), suggesting that the viral RNA may share a common origin to this highly conserved human RNA. Given the recently-discovered role of MALAT1 in controlling the metastasis of lung cancer cells (and its likely role in the proliferation of other cancer types), our structure provides insights that may enable the development of novel cancer therapies.