Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.The ability to monitor the identity and quantity of proteins that a cell produces would inform nearly all aspects of biology. Microarray-based measurements of mRNA abundance have revolutionized the study of gene expression (1). However, for several reasons there is a critical need for techniques that directly monitor protein synthesis. First, mRNA levels are an imperfect proxy for protein production because mRNA translation is subject to extensive regulation (2-4). Second predicting the exact protein product from the transcript sequence is not possible because of effects such as internal ribosome entry sites, initiation at non-AUG codons, and nonsense read-through (5,6). Finally, programmed ribosomal pausing during protein synthesis is thought to aid the cotranslational folding and secretion of some proteins (7-9).Polysome profiling, in which mRNAs are recovered from translating ribosomes for subsequent microarray analysis, can provide a useful estimate of protein synthesis (10). However, this approach suffers from limited resolution and accuracy. Additionally, upstream open reading frames (uORFs)-short translated sequences found in the 5′ untranslated region (5′UTR) of many genes-result in ribosomes that are bound to an mRNA and yet are not translating the encoded gene (11). Advances in quantitative proteomics circumvent some of these problems (2,3), but there currently are substantial limits on their ability to independently determine protein sequences and measure low-abundance proteins.The position of a translating ribosome can be precisely determined by using the fact that a ribosome protects a discrete footprint [∼30 nucleotides (nt)] on its mRNA template from nuclease digestion (12). We reasoned that advances in deep-sequencing technology, which NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript make it possible to read tens of millions of short (∼35 base pairs) DNA sequences in parallel (13), would allow the full analysis of ribosome footprints from cells. Here, we present a ribosome-...

The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.