Ribosome Binding Sites (RBSs)

Protein synthesis is regulated by the sequence and structure of the 5' untranslated region (UTR) of the mRNA
transcript. In prokaryotes, the ribosome binding site (RBS), which promotes efficient and accurate translation of
mRNA, is called the Shine-Dalgarno sequence after the scientists that first described it. This purine-rich sequence of
5' UTR is complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located within the 30S small
ribosomal subunit). Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs (see Figure 1 for the
consensus sequence). These sequences lie about 10 nucleotides upstream from the AUG start codon. Activity of a
RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator
AUG.

In eukaryotes, the Kozak sequence A/GCCACCAUGG, which lies within a short 5' untranslated region, directs
translation of mRNA. An mRNA lacking the Kozak consensus sequence may be translated efficiently in Ambion's in
vitro systems if it possesses a moderately long 5' UTR that lacks stable secondary structure. Our data demonstrate
that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic
ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal
binding sites.

Consensus RBS Sequences

Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of
fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient
translation.

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)Bernstein. 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 Laursen. 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 Gold. 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
bindingdeSmit. 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)Ringquist.

Four RBSs are shown. The first three all have the same aligned spacing (6 nucleotides). The fourth RBS has an
aligned spacing of 5 nucleotides since the portion of the sequence that matches the Shine-Dalgarno sequence is
shifted one nucleotide closer to the start codon than the other RBSs.

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 Chen.

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 bindingBoni.

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 paperIsaacs.

mRNA stability

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
synthesizedSmolke.