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Nicholas T Ingolia - One of the best experts on this subject based on the ideXlab platform.
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Ribosome Profiling: Global Views of Translation.
Cold Spring Harbor perspectives in biology, 2019Co-Authors: Nicholas T Ingolia, Jeffrey A. Hussmann, Jonathan S WeissmanAbstract:The translation of messenger RNA (mRNA) into protein and the folding of the resulting protein into an active form are prerequisites for virtually every cellular process and represent the single largest investment of energy by cells. Ribosome Profiling-based approaches have revolutionized our ability to monitor every step of protein synthesis in vivo, allowing one to measure the rate of protein synthesis across the proteome, annotate the protein coding capacity of genomes, monitor localized protein synthesis, and explore cotranslational folding and targeting. The rich and quantitative nature of Ribosome Profiling data provides an unprecedented opportunity to explore and model complex cellular processes. New analytical techniques and improved experimental protocols will provide a deeper understanding of the factors controlling translation speed and its impact on protein function and cell physiology as well as the role of ribosomal RNA and mRNA modifications in regulating translation.
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Transcriptome-wide measurement of translation by Ribosome Profiling.
Methods (San Diego Calif.), 2017Co-Authors: Nicholas J. Mcglincy, Nicholas T IngoliaAbstract:Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose amino acid sequence corresponds to the codon sequence of an mRNA's ORF. Translation is performed by the Ribosome; therefore, in order to understand translation and its regulation we must be able to determine the numbers and locations of Ribosomes on mRNAs in vivo. Furthermore, we must be able to examine their redistribution in different physiological contexts and in response to experimental manipulations. The Ribosome Profiling method provides us with an opportunity to learn these locations, by sequencing a cDNA library derived from the short fragments of mRNA covered by the Ribosome. Since its original description, the Ribosome Profiling method has undergone continuing development; in this article we describe the method's current state. Important improvements include: the incorporation of sample barcodes to enable library multiplexing, the incorporation of unique molecular identifiers to enable to removal of duplicated sequences, and the replacement of a gel-purification step with the enzymatic degradation of unligated linker.
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Ribosome Profiling as a Tool to Decipher Viral Complexity
Annual review of virology, 2015Co-Authors: Noam Stern-ginossar, Nicholas T IngoliaAbstract:Viral genomes harbor a variety of unusual translational phenomena that allow them to pack coding information more densely and evade host restriction mechanisms imposed by the cellular translational apparatus. Annotating translated sequences within these genomes thus poses particular challenges, but identifying the full complement of proteins encoded by a virus is critical for understanding its life cycle and defining the epitopes it presents for immune surveillance. Ribosome Profiling is an emerging technique for global analysis of translation that offers direct and experimental annotation of viral genomes. Ribosome Profiling has been applied to two herpesvirus genomes, those of human cytomegalovirus and Kaposi's sarcoma-associated herpesvirus, revealing translated sequences within presumptive long noncoding RNAs and identifying other micropeptides. Synthesis of these proteins has been confirmed by mass spectrometry and by identifying T cell responses following infection. Ribosome Profiling in other viruses will likely expand further our understanding of viral gene regulation and the proteome.
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Ribosome Profiling: new views of translation, from single codons to genome scale
Nature reviews. Genetics, 2014Co-Authors: Nicholas T IngoliaAbstract:Ribosome Profiling is a recently developed technique that uses deep sequencing to study translation in vivo. This approach has provided new insights into the identities and amounts of proteins produced by cells, as well as into the mechanism of protein synthesis itself.
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Genome-wide annotation and quantitation of translation by Ribosome Profiling.
Current protocols in molecular biology, 2013Co-Authors: Nicholas T Ingolia, Gloria A Brar, Silvia Rouskin, Anna M Mcgeachy, Jonathan S WeissmanAbstract:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. A protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing is presented here. This Ribosome-Profiling approach maps the exact positions of Ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating Ribosomes is described. The protocol described requires 5 to 7 days to generate a completed Ribosome Profiling sequencing library. Sequencing and data analysis requires an additional 4 to 5 days.
Jonathan S Weissman - One of the best experts on this subject based on the ideXlab platform.
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Ribosome Profiling: Global Views of Translation.
Cold Spring Harbor perspectives in biology, 2019Co-Authors: Nicholas T Ingolia, Jeffrey A. Hussmann, Jonathan S WeissmanAbstract:The translation of messenger RNA (mRNA) into protein and the folding of the resulting protein into an active form are prerequisites for virtually every cellular process and represent the single largest investment of energy by cells. Ribosome Profiling-based approaches have revolutionized our ability to monitor every step of protein synthesis in vivo, allowing one to measure the rate of protein synthesis across the proteome, annotate the protein coding capacity of genomes, monitor localized protein synthesis, and explore cotranslational folding and targeting. The rich and quantitative nature of Ribosome Profiling data provides an unprecedented opportunity to explore and model complex cellular processes. New analytical techniques and improved experimental protocols will provide a deeper understanding of the factors controlling translation speed and its impact on protein function and cell physiology as well as the role of ribosomal RNA and mRNA modifications in regulating translation.
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Abstract IA20: Monitoring translation in space and time with Ribosome Profiling
Innovating High-Throughput Technologies to Decode the Translational and RNA Structural Landscape of Cancer Cells, 2017Co-Authors: Jonathan S WeissmanAbstract:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on Ribosome Profiling (deep sequencing of Ribosome protected fragments) that dramatically expand our ability to follow translation in vivo. I will present recent applications of our Ribosome Profiling approach including the following: (1) Development of Ribosome Profiling protocols for a wide variety of eukaryotic and prokaryotic organisms. (2) Uses of Ribosome Profiling to globally monitor when chaperones, targeting factors or processing enzymes engage nascent chains. (3) Application of Ribosome Profiling to define the protein coding potential of complex genomes. (4) The development of a proximity-specific Ribosome Profiling assay for monitoring subcellular localized translation and its use to follow translation on the surface of the endoplasmic reticulum and mitochondria. Citation Format: Jonathan Weissman. Monitoring translation in space and time with Ribosome Profiling. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA20.
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Globally monitoring protein synthesis in time and space through Ribosome Profiling
The FASEB Journal, 2016Co-Authors: Jonathan S WeissmanAbstract:The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on Ribosome Profiling (deep sequencing of r...
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Ribosome Profiling of the Caulobacter Cell-Cycle
Biophysical Journal, 2014Co-Authors: Jared M Schrader, Jonathan S Weissman, Bo Zhou, Lucy ShapiroAbstract:An important goal in understanding cellular differentiation is to determine how the genetic information encoded in the genome is expressed properly in time and space to yield cell types with distinct functions. The bacterium Caulobacter crescentus has proven to be a valuable model organism for studying cellular differentiation processes that occur as a function of the cell cycle. Each Caulobacter cell division is asymmetric, yielding daughter cells with different cell fates. This process requires rapid and specific changes in gene expression during the cell cycle that are controlled at many levels, including transcriptional regulation, transient DNA methylation, differential proteolysis, and protein phospho-signaling. However, relatively little is known about the cell cycle control of mRNA translation. To understand the role of translational regulation in Caulobacter differentiation and asymmetric cell division, we are employing Ribosome Profiling to monitor genome-wide changes in translation at multiple times during cell cycle progression. Arrested Ribosomes are treated with ribonuclease to partially digest the mRNA, yielding short fragments protected by monoRibosomes. These Ribosome-protected mRNA fragments are purified and prepared for high throughput sequencing to map the position of cellular Ribosomes and quantify levels of translation. By comparing Ribosome Profiling levels with RNA-seq measurements of mRNA levels we find many new cases of genes whose translation only occurs during the proper phase of the cell cycle. We find that a majority of ORFs have a >2-fold change in translation efficiency during the cell cycle. This suggests that Caulobacter uses cell cycle-specific regulation of translation to ensure proper timing of gene expression.
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RRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in Ribosome Profiling experiments
Bioinformatics (Oxford England), 2013Co-Authors: Patrick B F O'connor, Jonathan S Weissman, John F. Atkins, Pavel V. BaranovAbstract:Motivation: Ribosome Profiling is a new technique that allows monitoring locations of translating Ribosomes on mRNA at a whole transcriptome level. A recent Ribosome Profiling study demonstrated that internal Shine–Dalgarno (SD) sequences have a major global effect on translation rates in bacteria: Ribosomes pause at SD sites in mRNA. Therefore, it is important to understand how SD sites effect mRNA movement through the Ribosome and generation of Ribosome footprints. Results: Here, we provide evidence that in addition to pausing effect, internal SD sequences induce a caterpillar-like movement of mRNA through the Ribosome cavity. Once an SD site binds to the Ribosome, it remains attached to it while the Ribosome decodes a few subsequent codons. This leads to asymmetric progressive elongation of Ribosome footprints at the 3′-end. It is likely that internal SD sequences induce a pause not on a single, but on several adjacent codons. This finding is important for our understanding of mRNA movement through the Ribosome and also should facilitate interpretation of Ribosome Profiling data. Contact: moc.liamg@nap.lavo.evarb
Allen R. Buskirk - One of the best experts on this subject based on the ideXlab platform.
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Protocol for Ribosome Profiling in Bacteria.
Bio-protocol, 2019Co-Authors: Fuad Mohammad, Allen R. BuskirkAbstract:Ribosome Profiling provides information on the position of Ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, Ribosome Profiling involves the isolation and sequencing of Ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of Ribosome Profiling in bacteria has been problematic, we report here a systematically optimized protocol for E. coli that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high magnesium concentration without the use of antibiotics. These improvements eliminate artifacts induced by harvesting cells by centrifugation or filtration and by use of chloramphenicol to arrest translation. These improvements are especially appropriate for studies where the exact position of the Ribosome is critical, and not merely the number of Ribosomes per message, such as studies aimed at monitoring differences in local elongation rates.
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A systematically-revised Ribosome Profiling method for bacteria reveals pauses at single-codon resolution
eLife, 2019Co-Authors: Fuad Mohammad, Rachel Green, Allen R. BuskirkAbstract:In eukaryotes, Ribosome Profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare Profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of Ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial Ribosome Profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.
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A Ribosome Profiling study of mRNA cleavage by the endonuclease RelE
Nucleic acids research, 2016Co-Authors: Jae-yeon Hwang, Allen R. BuskirkAbstract:Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the Ribosome. We obtained a global view of the effect of RelE on translation by Ribosome Profiling, observing that Ribosomes accumulate on the 5'-end of genes through dynamic cycles of mRNA cleavage, Ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promise as a method for generating Ribosome footprints. In bacteria, Profiling studies have suffered from relatively low resolution and have yielded no information on reading frame due to problems inherent to MNase digestion, the method used to degrade unprotected regions of mRNA. In contrast, we find that RelE yields precise 3'-ends that for the first time reveal reading frame in bacteria. Given that RelE has been shown to function in all three domains of life, RelE has potential to improve reading frame and shed light on A-site occupancy in Ribosome Profiling experiments more broadly.
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Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling
Cell reports, 2016Co-Authors: Fuad Mohammad, Christopher J. Woolstenhulme, Rachel Green, Allen R. BuskirkAbstract:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with Ribosome Profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the Profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long Ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by Ribosome Profiling.
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High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP
Cell reports, 2015Co-Authors: Christopher J. Woolstenhulme, Rachel Green, Nicholas R. Guydosh, Allen R. BuskirkAbstract:Ribosome Profiling is a powerful method for globally assessing the activity of Ribosomes in a cell. Despite its application in many organisms, Ribosome Profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli Profiling data, enabling us to more accurately assess Ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced Ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain.
Pavel V. Baranov - One of the best experts on this subject based on the ideXlab platform.
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Computational methods for Ribosome Profiling data analysis
Wiley interdisciplinary reviews. RNA, 2019Co-Authors: Stephen J Kiniry, Audrey M. Michel, Pavel V. BaranovAbstract:Since the introduction of the Ribosome Profiling technique in 2009 its popularity has greatly increased. It is widely used for the comprehensive assessment of gene expression and for studying the mechanisms of regulation at the translational level. As the number of Ribosome Profiling datasets being produced continues to grow, so too does the need for reliable software that can provide answers to the biological questions it can address. This review describes the computational methods and tools that have been developed to analyze Ribosome Profiling data at the different stages of the process. It starts with initial routine processing of raw data and follows with more specific tasks such as the identification of translated open reading frames, differential gene expression analysis, or evaluation of local or global codon decoding rates. The review pinpoints challenges associated with each step and explains the ways in which they are currently addressed. In addition it provides a comprehensive, albeit incomplete, list of publicly available software applicable to each step, which may be a beneficial starting point to those unexposed to Ribosome Profiling analysis. The outline of current challenges in Ribosome Profiling data analysis may inspire computational biologists to search for novel, potentially superior, solutions that will improve and expand the bioinformatician's toolbox for Ribosome Profiling data analysis. This article is characterized under: Translation > Ribosome Structure/Function RNA Evolution and Genomics > Computational Analyses of RNA Translation > Translation Mechanisms Translation > Translation Regulation.
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Illuminating translation with Ribosome Profiling spectra
Nature methods, 2016Co-Authors: Pavel V. Baranov, Audrey M. MichelAbstract:Software based on the spectral analysis of Ribosome Profiling improves the detection of translated segments in RNA molecules.
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surveying the relative impact of mrna features on local Ribosome Profiling read density in 28 datasets
bioRxiv, 2015Co-Authors: Patrick B F Oconnor, Dmitry E Andreev, Pavel V. BaranovAbstract:Ribosome Profiling is a promising technology for exploring gene expression. However, Ribosome Profiling data are characterized by a substantial number of outliers due to technical and biological factors. Here we introduce a simple computational method, Ribo-seq Unit Step Transformation (RUST) for the characterization of Ribosome Profiling data. We show that RUST is robust and outperforms conventional normalization techniques in the presence of sporadic noise. We used RUST to analyse 28 publicly available Ribosome Profiling datasets obtained from mammalian cells and tissues and from yeast. This revealed substantial protocol dependent variation in the composition of footprint libraries. We selected a high quality dataset to explore the mRNA features that affect local decoding rates and found that the amino acid identity encoded by the codon in the A-site is the major contributing factor followed by the identity of the codon itself and then the amino acid in the P-site. We also found that bulky amino acids slow down Ribosome movement when they occur within the peptide tunnel and Proline residues may decrease or increase Ribosome velocities depending on the context in which they occur. Moreover we show that a few parameters obtained with RUST are sufficient for predicting experimental densities with high accuracy. Due to its robustness and low computational demand, RUST could be used for quick routine characterization of Ribosome Profiling datasets to assess their quality as well as for the analysis of the relative impact of mRNA sequence features on local decoding rates.
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Ribosome Profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale.
Wiley interdisciplinary reviews. RNA, 2013Co-Authors: Audrey M. Michel, Pavel V. BaranovAbstract:Ribosome Profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of Ribosome protected mRNA fragments allowing the measurement of Ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of Ribosome density on individual RNAs. Since its invention, the Ribosome Profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original Ribosome Profiling protocol for studying specific aspects of translation. Ribosome Profiling of initiating Ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome Profiling of elongating Ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and Ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so Ribosome Profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. WIREs RNA 2013, 4:473–490. doi: 10.1002/wrna.1172 For further resources related to this article, please visit the WIREs website.
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RRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in Ribosome Profiling experiments
Bioinformatics (Oxford England), 2013Co-Authors: Patrick B F O'connor, Jonathan S Weissman, John F. Atkins, Pavel V. BaranovAbstract:Motivation: Ribosome Profiling is a new technique that allows monitoring locations of translating Ribosomes on mRNA at a whole transcriptome level. A recent Ribosome Profiling study demonstrated that internal Shine–Dalgarno (SD) sequences have a major global effect on translation rates in bacteria: Ribosomes pause at SD sites in mRNA. Therefore, it is important to understand how SD sites effect mRNA movement through the Ribosome and generation of Ribosome footprints. Results: Here, we provide evidence that in addition to pausing effect, internal SD sequences induce a caterpillar-like movement of mRNA through the Ribosome cavity. Once an SD site binds to the Ribosome, it remains attached to it while the Ribosome decodes a few subsequent codons. This leads to asymmetric progressive elongation of Ribosome footprints at the 3′-end. It is likely that internal SD sequences induce a pause not on a single, but on several adjacent codons. This finding is important for our understanding of mRNA movement through the Ribosome and also should facilitate interpretation of Ribosome Profiling data. Contact: moc.liamg@nap.lavo.evarb
Rachel Green - One of the best experts on this subject based on the ideXlab platform.
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high resolution Ribosome Profiling defines discrete Ribosome elongation states and translational regulation during cellular stress
Molecular Cell, 2019Co-Authors: Rachel Green, Colin Chihchien Wu, Boris Zinshteyn, Karen A WehnerAbstract:Summary Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define Ribosome conformational states corresponding to differently sized Ribosome-protected mRNA fragments (RPFs) generated by Ribosome Profiling. We show, using various genetic and environmental perturbations, that short 20–22 or classical 27–29 nucleotide RPFs correspond to Ribosomes with open or occupied ribosomal A sites, respectively. These distinct states of translation elongation are readily discerned by Ribosome Profiling in all eukaryotes we tested, including fungi, worms, and mammals. This high-resolution Ribosome Profiling approach reveals mechanisms of translation-elongation arrest during distinct stress conditions. Hyperosmotic stress inhibits translocation through Rck2-dependent eEF2 phosphorylation, whereas oxidative stress traps Ribosomes in a pre-translocation state, independent of Rck2-driven eEF2 phosphorylation. These results provide insights and approaches for defining the molecular events that impact translation elongation throughout biology.
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A systematically-revised Ribosome Profiling method for bacteria reveals pauses at single-codon resolution
eLife, 2019Co-Authors: Fuad Mohammad, Rachel Green, Allen R. BuskirkAbstract:In eukaryotes, Ribosome Profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare Profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of Ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial Ribosome Profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.
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Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling
Cell reports, 2016Co-Authors: Fuad Mohammad, Christopher J. Woolstenhulme, Rachel Green, Allen R. BuskirkAbstract:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with Ribosome Profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the Profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long Ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by Ribosome Profiling.
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High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP
Cell reports, 2015Co-Authors: Christopher J. Woolstenhulme, Rachel Green, Nicholas R. Guydosh, Allen R. BuskirkAbstract:Ribosome Profiling is a powerful method for globally assessing the activity of Ribosomes in a cell. Despite its application in many organisms, Ribosome Profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli Profiling data, enabling us to more accurately assess Ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced Ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain.
