Ribosome Binding Sites/Design
Design aspects of prokaryotic Ribosome Binding Sites (RBSs)
An RBS is an RNA sequence upstream of the start codon that affects the rate at which a particular Open Reading Frame (ORF) is translated. Various aspects of RBS design affect the rate at which the ORF is translated. This page describes some of those design aspects. You can use this informtation either to better understand how RBSs work, or to design new RBSs. Before designing a new RBS, you should first see if you can use some of the existing high quality RBSs. This is because we think it is most useful to the community to develop a small number of very-well characterized collection of RBS rather than lots of rarely used RBSs.
If you'd like an introduction to the basics of how prokaryotic RBSs work, see here. That page also discusses the consensus RBS sequence for E. coli. An important fact to keep in mind about RBS design and function is that that the RBS functions in a larger context. For example, the RBS can interact with sequences upstream (such as sequence transcribed as part of the promoter, or an upstream ORF). The RBS also functions in context with downstream sequence, for example the ribosome binds jointly to the RBS and start codon at about the same time. Hence, when analyzing or designing an RBS, the surrounding sequence must always be considered. We'll see some examples of that below.
The RBS affects the translation rate of an ORF in two main ways - i) the rate at which ribosomes are recruited to the mRNA and initiate translation is dependent on the sequence of the RBS, and ii) the RBS can also affect the stability of the mRNA, thereby affecting the number of proteins made over the lifetime of the mRNA (the half-life of an mRNA is often less than 5 minutes). Neither of these mechanisms is completely understood but what we do know is discussed below.
Translation initiation rate
Translation initiation in prokaryotes is a complex process involving the ribosome, the mRNA, and several other proteins (initiation factors). For a good review, see this paper by Laursen et al . Very crudely, we can break translation initiation down into two major steps - i) binding of the ribosome and associated factors to the mRNA, and ii) conversion of the bound ribosome into a translating ribosome lengthening processing along the mRNA. The rate of the first step can be increased by making the RBS highly complementary to the free end of the 16s rRNA (see here for an introduction to how ribosomes bind to mRNA) and by ensuring that the start codon is AUG . The rate of ribosome binding rate can also be increased by ensuring that there is minimal secondary structure in the neighborhood of the RBS. Since binding between the RBS and the ribosome is mediated by base-pairing interactions, competition for the RBS from other sequences on the mRNA, can reduce the rate of ribosome binding. The rate of the second step in translation initiation, conversion of the bound ribosome into an initiation complex is dependent on the spacing between the RBS and the start codon being optimal (5-6bp).
RBSs will commonly include only a portion of the Shine-Dalgarno sequence. When looking at the spacing between the RBS and the start codon, it is important to think of the aligned spacing rather than just the absolute spacing. The idea of an aligned spacing is illustrated in the figure on the right. In essence, if only a portion of the Shine-Dalgarno sequence is included in the RBS, the spacing that matters is between wherever the center of the full Shine-Dalgarno sequence would be and the start codon rather than between the included portion of the Shine-Dalgarno sequence and the start codon .
While the Shine-Dalgarno portion of the RBS is critical to the strength of the RBS, the sequence upstream of the Shine-Dalgarno sequence is also important. One of the ribosomal proteins, S1, is known to bind to adenine bases upstream from the Shine-Dalgarno sequence. As a result, the RBS can be made stronger by adding more adenines in the sequence upstream of the RBS. Note that the promoter may add some bases onto the start of the mRNA that may affect the strength of the RBS by affecting S1 binding.
As discussed above, the degree of secondary structure can affect the translation initiation rate. This fact can be used to produce regulated translation initiation rates. For an introduction to this technology (known as riboregulation), see this page and this recent paper.
In addition to affecting the translation rate per unit time, the Ribosome Binding Site affects the level of protein synthesis in a second way. That is because the stability of the mRNA affects the steady state level of mRNA; a stable mRNA will have a higher steady state level than an unstable mRNA that is being produced as an identical rate. Since the primary sequence and the secondary structure of an RBS (for example, the RBS could introduce a RNase site) can affect the stability of the mRNA, the RBS can affect the amount of mRNA and hence the amount of protein that is synthesized.
Are you ready to use all this information to design a new RBS? We have a tutorial on how to turn your design into physical DNA.