Translational control of fis
- Author(s): Nafissi, Maryam
- Advisor(s): Johnson, Reid C
- et al.
In order to translate an mRNA into a protein, the ribosome has to proceed through initiation, elongation, and termination steps guided by mRNA signals and auxiliary protein factors. Translation initiation is highly regulated in prokaryotes and requires the step-wise assembly of ribosomal subunits, mRNA, initiator tRNA, and initiation factors. Even though binding between the 16S rRNA (anti-Shine Dalgarno) and its complementary sequence on the mRNA (Shine Dalgarno) is thought to be the major interaction driving translation initiation, additional cis- or trans-acting factors can also provide control. In the current work, we describe novel mRNA elements important for efficient translation initiation of the fis mRNA, which encodes a highly abundant Escherichia coli DNA binding protein. The fis mRNA is part of a bicistronic message that contains dusB at the 5f end encoding a tRNA modifying enzyme. Even though the cellular mRNA levels are comparable, Fis protein levels are much higher than DusB. Moreover, the translation of the downstream fis coding region does not depend on translation of dusB. Rather than relying on a SD sequence, fis translation requires upstream RNA elements located within the 3Oe end of dusB. These noncanonical elements include a conserved AU-rich sequence (AU) and a putative RNA stem-loop structure (SL). We propose that the AU element is involved in recruiting the 30S ribosome to the translation start site, whereas the SL indirectly enhances translation initiation via an anti-inhibitory mechanism. Previous studies have shown that cellular Fis levels are determined by multiple mechanisms of transcriptional regulation, and the present work now demonstrates that multiple and unusual mechanisms of translational control are also operating.
Fis functions as a global transcriptional regulator and has also been proposed to play a role in chromosome organization and compaction. To investigate Fis binding to the E. coli chromosome in vivo, genome-wide ChIP-chip binding studies were performed under different growth and mutant conditions. Fis was found to bind prolifically throughout the chromosome, and as expected, genome-wide Fis binding decreases under growth conditions that reduce fis expression. Surprisingly, Fis binding was found not to be generally influenced by other abundant nucleoid-associated proteins like HU and H-NS. We analyzed Fis binding in vitro to subclasses of Fis binding regions identified by the ChIP-chip profiles. Narrow Fis binding peaks (<500 bp) were found to primarily consist of a single high-affinity Fis binding site in vitro whereas broad binding peaks (>1.5 kb) often contained 3-4 localized high-affinity Fis binding sites. These broad Fis binding regions are good candidates for chromosome organizing centers. In summary, work in this thesis advances our understanding of noncanonical translation initiation in prokaryotes and the targeted binding of the nucleoid-associated protein Fis to the E. coli chromosome.