Identifying A- and P-site locations on ribosome-protected mRNA fragments using Integer Programmering Nabeel Ahmed, Pietro Sormanni, Abstract
Identifying
the A- and P-site locations on ribosome-protected mRNA fragments from
Ribo-Seq experiments is a fundamental step in the quantitative analysis
of transcriptome-wide translation properties at the codon level. Many
analyses of Ribo-Seq data have utilized heuristic approaches applied to a
narrow range of fragment sizes to identify the A-site. In this study,
we use Integer Programming to identify the A-site by maximizing an
objective function that reflects the fact that the ribosome’s A-site on
ribosome-protected fragments must reside between the second and stop
codons of an mRNA. This identifies the A-site location as a function of
the fragment’s size and its 5′ end reading frame in Ribo-Seq data
generated from S. cerevisiae and mouse embryonic stem cells. The
correctness of the identified A-site locations is demonstrated by
showing that this method, as compared to others, yields the largest
ribosome density at established stalling sites. By providing greater
accuracy and utilization of a wider range of fragment sizes, our
approach increases the signal-to-noise ratio of underlying biological
signals associated with translation elongation at the codon length
scale.
P-SITE:
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/p-site
Molecular Motors and MotilityB.S. Cooperman, in Comprehensive Biophysics, 2012 4.21.5.1.2 Downstream mRNA 2° structure
Because translation requires that mRNA present at the A, P, and E sites be in single-strand form, 2° structures such as stem loops or pseudoknots that occur downstream from the decoding center, and that need to be unraveled before translation, can modulate the rate of protein synthesis.78,83–86 Transcription and translation are tightly coupled in prokaryotes with initiation of translation occurring shortly after transcription initiation. Given this tight coupling and the close spacing between ribosomes on a polysome, the mRNA length available for secondary structure formation during in vivo elongation is typically quite limited.
E. coli ribosomes on polysomes engaged in cell-free protein synthesis have an average spacing of 97 nts87 (suggesting an average gap between ribosomes of ∼60 nts), a value that is not dissimilar from the average value of one ribosome/156 nts found in the polysomes of yeast cells.88
However, longer lengths may sometimes become available (a) when
transcription and translation are transiently decoupled, or (b)
downstream from a paused or stalled ribosome within a polysome.89
During elongation, 2° structure is presumably unwound by an intrinsic
helicase activity of the ribosome, which is believed to act at nucleotide +11, where the 5′-end of the codon at the P-site is indexed as +1.90,91
Eukaryotic Protein Biosynthesis: The Elongation CycleA.A. Komar, ... W.C. Merrick, in Encyclopedia of Biological Chemistry (Second Edition), 2013
Influence of Other Sites
In the discussion above, reference was made to an (exit) E site (see also Figure 3). This site, to the 5′ side of the P site, has been visualized by X-ray crystallography (in prokaryotes), cryo-electron microscopy (in both pro- and eukaryotes) and inferred by nuclease
footprinting. The most important effect of the E site is that it
appears to increase the stringency for the correct matching of the codon and anticodon
in the A site of the ribosome. It should be noted that for the original
binding of the first aa-tRNA, Met-tRNAi, the E site was unoccupied. One
might anticipate that this would allow for less stringent recognition
of the initiating AUG codon; however, this strict recognition appears to
result from the unique set of factors associated with the initiation
process. A second proposed site is the F site or entry site which is
just 3′ of the A site. This may be an initial test-binding site (check
of codon/anticodon match) such that correctly matching complexes would
be pulled into the A site while incorrectly matching complexes would
dissociate. The advantage of an F site would be that it would be more
accessible to ternary complexes
of eEF1A·GTP·aa-tRNA as both the A site and the P site appear to be
partially occluded at the interface of the large and small ribosomal
subunits. However, the biochemical evidence supporting the existence of
the F site is weak compared to that for the E site.
F-SITE Entry site
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