Absolute quantification of translational regulation and burden using combined sequencing approaches

Abstract

1Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop 2 codons provide signals to initiate and terminate translation, while stable secondary mRNA structures 3 can induce translational recoding events. Fluorescent proteins are commonly used to characterize 4 such elements but require the modification of a part's natural context and allow only a few 5 parameters to be monitored concurrently. Here, we develop an approach that combines ribosome 6 profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput 7 characterization of genetic parts controlling translation in absolute units. We simultaneously 8 measure 743 translation initiation rates and 746 termination efficiencies across the Escherichia coli 9 transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot 10 structure. By analyzing the transcriptional and translational response, we discover that sequestered 11 ribosomes at the pseudoknot contribute to a σ 32 -mediated stress response, codon-specific pausing, 12 and a drop in translation initiation rates across the cell. Our work demonstrates the power of 13 integrating global approaches towards a comprehensive and quantitative understanding of gene 14 regulation and burden in living cells. 15 42 2016). When used for characterization, the part of interest is usually placed into a new genetic 43 backbone (often a plasmid) and its behavior is directly linked to the expression of one or more 44 fluorescent proteins (Cambray et al, 2013). When debugging a circuit failure, it is not possible to 45 extract the part of interest as the context of the circuit is important. For circuits that use transcription 46 rate (i.e. RNAP flux) as a common signal between components (Canton et al, 2008), debugging 47 plasmids containing a promoter responsive to the signal of interest have been used to track the 48 propagation of signals and reveal the root cause of failures (Nielsen et al, 2016). Alternatively, any 49 4genes whose expression is controlled by the part of interest can be tagged by a fluorescent protein 50 (Snapp, 2005). Such modifications allow for a readout of protein level but come at the cost of 51 alterations to the circuit. This is problematic as there is no guarantee the fluorescent tag itself will not 52 affect a part's function (Baens et al, 2006; Margolin, 2012). 53 The past decade has seen tremendous advances in sequencing technologies. This has 54 resulted in continuously falling costs and a growing range of information that can be captured 55 (Goodwin et al, 2016). Sequencing methods exist to measure chromosomal architecture 56 (Lieberman-aiden et al, 2009), RNA secondary structure (Lucks et al, 2011), DNA and RNA 57 abundance (Conesa et al, 2016), and translation efficiency (Ingolia, 2014). New developments have 58 expanded the capabilities even further towards more quantitve measurements of transcription and 59 protein synthesis rates with native elongating transcript sequencing (NET-seq) (Maye...

Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology

“…Figure 2 shows the resulting fraction of co-translated dimerisation (α) in relation to key model parameters (Figure 2A): the binding rate of the ribosomes (λ), the length of the monomers (measured in amino acids) and the reaction parameter (k D ). These results are shown for two different values of the reaction parameter (dimensionless), 1 ( Figure 2B) and 10 ( Figure 2C), at experimentally obtained values for the binding rate [43], λ ≈ 0.1s − 1. As observed here, α is minimum (from 0 to ≈0.2) at lower values of λ and protein length; although λ is the limiting rate here, since at very low values the protein length does not make a difference.…”

Contemporary Tools for Regulating Gene Expression in Bacteria

“…Communal synthetic biology resources and large collaboration, such as iGEM [40], demonstrate the power of interoperability and sharing of standard DNA parts. As we move towards more complex circuit characterisation technologies (i.e., for quantification of transcription and translation [9,41,42]), the use of standards for designing, executing, measuring, and logging experimental efforts will become yet more essential.…”

“…There is a cellular burden associated with hosting heterologous circuits [7][8][9]. Circuit-associated burden is an important consideration that must be accounted for as we try to build genetic circuits of increasing complexity.…”

“…Given developments in burden quantification and the increasing complexity of synthetic gene regulatory networks, our ability to predictably engineer biology is increasing [7,9,10]. The burden associated with heterologous circuit integration is often caused by a lack of orthogonality, competition for resources [8,11,12], such as ribosomes between heterologous and native systems [13][14][15], and even mRNA toxicity [16].…”

Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage

“…The level of gene expression can be calculated as the average profile height over its length, reported as FPKM (fragments per kilobase of transcript per million mapped reads). FPKM is an estimate of mRNA transcript copy number and has been converted to absolute units using RNA spike-in standards 7,37,38 . When there is a sharp increase or decrease in RNA-seq transcription profile, the magnitude can be used to infer the strength of a promoter or terminator, respectively [39][40][41][42][43][44][45][46][47] .…”