Illumina HiSeq 2500 (Saccharomyces cerevisiae) Zid BM, Guzikowski AR, Zhang J, Begovich K, Zhu S, Cohn MH cerevisiae in actue glucose starvation, glucose readdition, and mutli-day growth conditions MRNA and ribosome occupancy profiles of S. GTI-seq revealed a pronounced single peak highly focused at the annotated TIS with few reads recovered in the downstream region ( Figure 1C, left panel). Together, these results demonstrate how yeast regulate translation in response to glucose starvation. As we reported previously, LTM-based ribosome profiling (GTI-seq) offers global mapping of translation initiation sites (TIS) at single-nucleotide resolution 19. We used reporter assays to corroborate these findings in vivo. Further, it also indicated ribosomes previously built up can resume translation upon glucose readdition. Our ribosome profiling analysis suggested that elongation slows during starvation which leads to a buildup of ribosomes on the 3’ halves of mRNAs. Additionally, we performed ribosome profiling in samples that had glucose added back following acute starvation and observed a wave of new ribosome movement near the start codon and approximately 2,000 nucleotides downstream on open reading frames after one and five minutes of readdition, respectively. This is consistent with a read density analysis which demonstrated increased density on the 3’ half of genes after glucose starvation. In acute starvation, polarity shifted positive at our earliest time points but did not continue to do so at later time points. We found that ribosome polarity became positive in postdiauxic shift conditions relative to log phase. We profiled a time course of acute glucose starvation followed by glucose readdition and a multi-day growth course through the diauxic shift into stationary phase. Based on cell typespecific epitope tagging with genetic tools, two closely related methods were developed, named TRAP (Translating Ribosome Affinity Purification) and RiboTag. Here, we used ribosome profiling to calculate the ribosome polarity scores and changes in ribosome footprint read density in both aggregate and gene-specific ways. A complementary approach overcoming some of these limitations is the isolation of ribosome-associated mRNAs from genetically defined cell populations. Ribosome profiling provides an opportunity to not only assess how the relative abundance of ribosome association with mRNAs changes in different conditions, but to look more closely at where along mRNAs those ribosomes bind. cerevisiaeĮxpression profiling by high throughput sequencing Ribosome profiling and RNA-seq of an acute glucose starvation timecourse and 5 day growth course in S. GEO help: Mouse over screen elements for information.

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