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In prokaryotic translation, how critical for efficient translation is the location of the ribosome binding site, relative to the start codon?

Ideally, it is supposed to be -7b away from the start. How about if it is -9 bases away or even more? Will this have an observable effect on translation?

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I found an oldish paper on this topic (from 1994). Here's a summary:

Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. by Chen, Bjerknes, Kumar, & Jay. Nucleic Acids Research. (1994)

Experiment

The authors constructed a series of synthetic RBS regions that varied the length separating a synthetic 5-nt Shine-Dalgarno sequence from the start codon. The regions varied in size between 2 to 17 nt. They assayed the activity of a downstream enzyme, chloramphenicol acetyltransferase.

Conclusion

The authors concluded that the optimal spacing between a consensus 5-nt Shine-Dalgarno sequence (5'-GAGGT-3') and the start site was 5 nt. Note: this synthetic SD was made of the last 5 nt of a 9 nt SD consensus sequence. They also tested a synthetic SD made from the first 5 nt (5'-TAAGG-3')of the consensus SD. In this case they found the optimal distance was 9 nt.

So the optimal distance depends on where your desired SD aligns with the consensus SD sequence, which optimally is 5 nt from the start. Read on for more details.

Details

  • the RBS is considered to be large, extending 20bp on either side of a core Shine-Dalgarno (SD) sequence. These days, I often hear of the RBS spoken of in sizes that are equivalent to the SD. So in the parlance of the paper, you question is rephrased as "how does distance of the SD from the start codon effect translation?"

  • the canonical SD sequence referenced in the paper is 5'-UAAGGAGGU-3'. It is 9 nucleotides long. Distances between the SD and the start codon are defined as the number of nucleotides separate the 3' Uracil of the SD from the Adenine of the start AUG.

  • Example: the distance is 5 nt in the following mRNA

           5'.....UAAGGAGGUnnnnnAUG......3'

  • if the SD is not a complete 9 nt long, the distance is between the position of where the canonical Uracil would occur. In the following example, the distance is still calculated as 5 nt:

           5'.....UAAGGAnnnnnnnnAUG......3'

  • the average distance between the SD sequence and the start codon varies considerably and on average is 7 nt. Other investigators have found "optimal" spacing (circa 1994) ranged from 5 to 13 nt.

  • the SD site complements with region on the 16s rRNA. The start codon complements to the anticodon of fMet-tRNA loaded into the ribosomal P-site. So there are two distinct sites on the ribosome that contact the mRNA during translation initiation.

Also good to read

Note

I enjoyed researching this question because I found my own knowledge to be lacking solid empirical details. I do not have any direct experience besides this little lit review with this topic.

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  • $\begingroup$ Thanks for your thorough answer. I guess I haven't realized that the RBS end is determined by this U base. Does this mean that it can be 100 bases away from ATG? I am actually asking this question, because I have a synthetic construct with RBS annotated on it and some of them are really far away from the start codon. Which could mean either wrong annotation of the following gene sequence, or that the distance doesn't matter that much. But on the other hand I thought that the ribosome should bind both the RBS and the start codon at the same time which I don't see possible with long sequences. $\endgroup$
    – Gergana Vandova
    Commented Dec 15, 2011 at 1:23
  • $\begingroup$ What I am also concerned about, are secondary structures formed between the RBS and the upsream or dowstream sequence. Any thoughts on that? Thanks again :-) $\endgroup$
    – Gergana Vandova
    Commented Dec 15, 2011 at 1:23
  • $\begingroup$ The main idea in the Chen paper I referenced was that the distance between the Shine-Dalgarno sequence / RBS and the start codon is limited by the physical distance between the complementary sites on the RBS. This distance seems to be about 5bp. According to this old paper, the ribosome should not be able to bind effectively at all if the RBS is farther than say 20bp from the start site because the ribosome can't stretch out far enough to reach both sites at the same time. New research may have more to say on this topic. $\endgroup$
    – Mac Cowell
    Commented Dec 15, 2011 at 1:29
  • $\begingroup$ Secondary structure has significant effect on translation efficiency. Many riboregulators have been observed and designed. For instance, one can design stem-loop secondary structure around the RBS region. The region will self-complement in the absence of a particular aptamer or a strong complement. In their presence, it unfolds and allows the ribosome access to the RBS and start site. Engineered riboregulators enable post-transcriptional control of gene expression. Isaccs et al, Nature, 2004. $\endgroup$
    – Mac Cowell
    Commented Dec 15, 2011 at 1:41
  • $\begingroup$ It was an interesting question to investigate and I enjoyed learning about it. I hope more well-posed and provocative questions are asked here and that users react to them by actually going out and reading up on the primary literature. Looks like that happens occasionally. Cool. $\endgroup$
    – Mac Cowell
    Commented Jan 24, 2012 at 8:16

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