Translation initiation complex formation during stress

To cope with an ever-changing environment, bacteria have developed a variety of adaptations to combat stress. One such post-transcriptional stress response in Escherichia coli is based on a ribosomal modification by the endonucleolytic activity of MazF, the toxin component of the toxin-antitoxin system mazEF. Stress-induced MazF activity leads to cleavage events at single-stranded ACA sequences resulting in bulk mRNA degradation. However, a subset of mRNA are only cleaved within their 5- untranslated region (5-UTR) resulting in shortened or leaderless mRNAs lacking canonical ribosome binding signals. Additionally, MazF removes the 3-terminus of the 16S rRNA containing the complementary mRNA recognition region. Thereby a sub-population of stress ribosomes, termed 70S43, are generated. MazF cleavage of both the mRNA and the ribosome allow translation to continue as an adaptation to the stress conditions. Surprisingly, besides removal of the mRNA 5-UTR, MazF cleavage also results in a hydroxyl group at the 5-terminus as opposed to the 5-triphosphate group that originates as a result of mRNA transcription. Although wild-type ribosomes are inefficient at binding mRNAs containing a 5-hydroxyl, 70S43 ribosomes do not appear to be negatively affected. Understanding the efficient translation of MazF-processed mRNA by 70S43 ribosomes is the goal of the proposed project entitled Insights into translation initiation complex formation by the specialized 70S43 ribosomes on MazF-processed mRNAs and a potential impact of EF-P. We hypothesize that the processing of the ribosome results in the loss of a discriminatory factor allowing the 70S 43 ribosome to promiscuously bind to the MazF-processed mRNAs. To analyze the impact of the 5-hydroxyl on ribosome binding, we will investigate the formation and stability of ribosome complexes on both 5- hydroxyl and 5-triphosphate mRNAs. We will also investigate if presence of a 5-hydroxyl has any effect on synthesis of the encoded protein. In addition, we will determine the role that the elongation factor, EF-P, has on the translation of MazF-processed mRNAs. I plan to employ primer extension inhibition assays, ternary complex stability assays, metabolic pulse labeling and RNAseq to address these questions. This proposed project explores the way the cell globally regulates protein production by modifying the translational machinery itself. Stress-induced ribosome heterogeneity illustrates the ability of the translational machinery to not only act as a protein factory but also to globally regulate gene expression.

Microorganisms are ubiquitous throughout nature and thus must be able to adapt to a variety of stressors in a manner that will ensure their survival. Stress response at the post-transcriptional level is especially effective as it allows for the bacteria to adjust its protein output without the time-consuming step of drastically altering the transcriptome. More specifically, the bacteria may even modify the ribosome, the protein factory within the cell, to enact global changes in the translatome. In this study, we furthered our understanding of the modified stress ribosome population in Escherichia coli and its mechanism for interaction with various mRNAs. We were able to show that the stressed ribosomes that contain a truncated 16S rRNA are more promiscuous and are able to bind to mRNAs containing either a 5'-triphosphate, representing de novo transcribed mRNAs, or a 5'-hydroxyl, representing mRNAs nucleolytically cleaved, which wild-type ribosomes are unable to successfully bind. Even though during this response, many mRNAs are cleaved and recycled, the residual mRNAs generally no longer contain sequences that will attenuate translation. Therefore, it appears that this stress response allows for uninhibited production of proteins essential to cell survival. Understanding the stress response will allow us to specifically target this mechanism rendering the bacteria unable to efficiently recover and survive any multitude of inflicted stressors. This study was also able to uncover additional functions for elongation factor P (EF-P), a translational protein known to help relieve stalled ribosomes at polyproline stretches. Our data suggests that the presence of EF-P negatively impacts translation of leaderless mRNAs. As this impact was even more pronounced during nutrient limiting growth conditions, in which elongation is slowed therefore the necessity to resolve stalled ribosomes by EF-P is relieved, it suggests that the impact of EF-P on leaderless translation is not related to elongation. Additionally, EF-P was shown to have nucleolytic function, similar to its archaeal homolog. The nuclease activity of EF-P was characterized, and its potential recognition sequence documented. Both the modified and unmodified versions of EF-P were able to cleave leadered and leaderless mRNAs as well as tRNA. However, the presence of ribosomes appears to mitigate the nuclease activity either by protecting the RNA from cleavage or acting as a sponge to bind EF-P keeping it from interacting with the RNA. Taken together, this data suggests that EF-P plays other roles in the cell, potentially even in the translational process, which need to be further explored.