If you look carefully at the following graph, you can see that the cloud of gold-colored data points (representing the compositional stats for the second base in codons of Clostridium botulinum) has a second, "breakaway" cloud underneath it. (See arrow.)
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| Codon base composition statistics for Clostridium botulinum. Notice the breakaway cloud of gold points under the main cloud (arrow). These points represent genes in which most codons have a pyrimidine in the middle base of each codon. |
To review: I made this plot by going through each DNA sequence for each coding ("CDS") gene of C. botulinum, and for each gene, I went through all codons and calculated the average purine content (as well as the average G+C content) of the bases at a positions one, two, and three in the codons. Thus, every dot represents the stats for one gene's worth of data.
After looking at graphs of this sort, three key facts about codon bases leap out:
- Most codons, for most genes, have a purine as the first base (notice how the red cloud of points is higher than the others, centering on y=0.7).
- The third base (often called the "wobble" base; shown blue here) has the most extreme G+C value. (This is well known.)
- The middle base falls in one of two positions (high or low) on the purine (y) axis. There's a primary cloud of data points and a secondary cloud in a distinct region below the main cloud. The secondary cloud of gold points is centered at about 0.3 on the y-axis, meaning these are genes in which the second codon base tends to be a pyrimidine.
If you examine the standard codon translation table (below), you can see that codons with a pyrimidine in the second position (represented by the first two columns of the table) code primarily for nonpolar amino acids. When a pyrimidine is in the second base, the possible amino acids are phenylalanine, serine, leucine, proline, isoleucine, methionine, threonine, valine, and alanine. Of these, all but serine and threonine are nonpolar. Therefore, a pyrimidine in position two of a codon means there's at least a 75% chance that the amino acid will have a nonpolar, hydrophobic side group.
Virtually all proteins contain some nonpolar amino acids, but when a protein contains mostly nonpolar amino acids, that protein is destined to wind up in the cell membrane (which is largely made up of lipids). Thus, we can expect to find that genes in the "breakaway" cloud of gold points in the graph further above represent membrane-associated proteins.
To check this hypothesis, I wrote a script that harvested the gene names, and protein-product names, of all the "breakaway cloud" data points. After purging genes annotated (unhelpfully) as "hypothetical protein," I was left with 37 "known" genes. They're shown in the following table.
| Gene | Product |
| CLC_0571 | arsenical pump family protein |
| CLC_1058 | L-lactate permease |
| CLC_1550 | carbohydrate ABC transporter permease |
| CLC_3115 | xanthine/uracil permease family protein |
| CLC_0813 | arsenical-resistance protein |
| CLC_1018 | putative anion ABC transporter, permease protein |
| CLC_1687 | xanthine/uracil permease family protein |
| CLC_3633 | sporulation integral membrane protein YtvI |
| CLC_2382 | phosphate ABC transporter, permease protein PstA |
| CLC_0189 | ZIP transporter family protein |
| CLC_3351 | sodium:dicarboxylate symporter family protein |
| CLC_0971 | cobalt transport protein CbiM |
| CLC_1534 | methionine ABC transporter permease |
| CLC_2798 | xanthine/uracil permease family protein |
| CLC_1397 | manganese/zinc/iron chelate ABC transporter permease |
| CLC_1836 | stage III sporulation protein AD |
| CLC_0528 | high-affinity branched-chain amino acid ABC transporter, permease protein |
| CLC_0430 | electron transport complex, RnfABCDGE type, A subunit |
| CLC_2523 | flagellar biosynthesis protein FliP |
| CLC_0401 | amino acid permease family protein |
| CLC_0383 | lrgB-like family protein |
| CLC_0457 | chromate transporter protein |
| CLC_0291 | sodium:dicarboxylate symporter family protein |
| CLC_0427 | electron transport complex, RnfABCDGE type, D subunit |
| CLC_1281 | putative transcriptional regulator |
| CLC_2008 | ABC transporter, permease protein |
| CLC_0868 | branched-chain amino acid transport system II carrier protein |
| CLC_1237 | monovalent cation:proton antiporter-2 (CPA2) family protein |
| CLC_1137 | methionine ABC transporter permease |
| CLC_0764 | putative drug resistance ABC-2 type transporter, permease protein |
| CLC_1953 | xanthine/uracil permease family protein |
| CLC_2444 | auxin efflux carrier family protein |
| CLC_0897 | putative ABC transporter, permease protein |
| CLC_1555 | C4-dicarboxylate transporter/malic acid transport protein |
| CLC_0374 | xanthine/uracil permease family protein |
| CLC_0470 | undecaprenyl pyrophosphate phosphatase |
| CLC_2648 | monovalent cation:proton antiporter-2 (CPA2) family protein |
Notice that with the exception of CLC_1281, a "putative transcriptional regulator," every gene product represents a membrane-associated protein: transporters, carrier proteins, permeases, etc.
I ran the same experiment on genes from Streptomyces griseus (strain XylbKG-1) and came up with 222 genes having high pyrimidine content in base two. All 222 genes specify membrane-associated proteins. (The full list is in a table below.)
The bottom line: Base two of codons acts as a binary switch. If the base is a pyrimidine, the associated amino acid will most likely (75% chance) be nonpolar. If the base is a purine, the codon will either be a stop codon (3 out of 32 codons) or the amino acid will be polar (26 out of 29 codons).
Here's the list of 222 genes from S. griseus in which the middle codon base is predominantly a pyrimidine:
| SACT1_0608 | ABC-type transporter, integral membrane subunit |
| SACT1_3730 | major facilitator superfamily MFS_1 |
| SACT1_4066 | cation efflux protein |
| SACT1_5911 | ABC-2 type transporter |
| SACT1_6577 | SNARE associated protein |
| SACT1_6966 | ABC-type transporter, integral membrane subunit |
| SACT1_7160 | major facilitator superfamily MFS_1 |
| SACT1_3151 | NADH-ubiquinone/plastoquinone oxidoreductase chain 6 |
| SACT1_3682 | drug resistance transporter, EmrB/QacA subfamily |
| SACT1_5431 | Citrate transporter |
| SACT1_3199 | proton-translocating NADH-quinone oxidoreductase, chain M |
| SACT1_7301 | putative integral membrane protein |
| SACT1_3198 | NAD(P)H-quinone oxidoreductase subunit 2 |
| SACT1_2008 | arsenical-resistance protein |
| SACT1_3149 | proton-translocating NADH-quinone oxidoreductase, chain L |
| SACT1_3967 | MATE efflux family protein |
| SACT1_3148 | proton-translocating NADH-quinone oxidoreductase, chain M |
| SACT1_5571 | major facilitator superfamily MFS_1 |
| SACT1_0651 | major facilitator superfamily MFS_1 |
| SACT1_2669 | ABC-type transporter, integral membrane subunit |
| SACT1_1805 | NADH dehydrogenase (quinone) |
| SACT1_3147 | NAD(P)H-quinone oxidoreductase subunit 2 |
| SACT1_6961 | major facilitator superfamily MFS_1 |
| SACT1_0992 | ABC-2 type transporter |
| SACT1_2619 | major facilitator superfamily MFS_1 |
| SACT1_0507 | major facilitator superfamily MFS_1 |
| SACT1_0649 | ABC-type transporter, integral membrane subunit |
| SACT1_0800 | glycosyl transferase family 4 |
| SACT1_1659 | ABC-type transporter, integral membrane subunit |
| SACT1_1803 | multiple resistance and pH regulation protein F |
| SACT1_4190 | putative ABC transporter permease protein |
| SACT1_5522 | drug resistance transporter, EmrB/QacA subfamily |
| SACT1_5568 | drug resistance transporter, EmrB/QacA subfamily |
| SACT1_7248 | Lysine exporter protein (LYSE/YGGA) |
| SACT1_0266 | ABC-2 type transporter |
| SACT1_0847 | Na+/solute symporter |
| SACT1_4378 | ABC-type transporter, integral membrane subunit |
| SACT1_6766 | ABC-type transporter, integral membrane subunit |
| SACT1_2522 | putative integral membrane protein |
| SACT1_4762 | amino acid permease-associated region |
| SACT1_4901 | major facilitator superfamily MFS_1 |
| SACT1_2616 | multiple antibiotic resistance (MarC)-related protein |
| SACT1_3961 | major facilitator superfamily MFS_1 |
| SACT1_6236 | MIP family channel protein |
| SACT1_1319 | protein of unknown function UPF0016 |
| SACT1_2332 | copper resistance D domain protein |
| SACT1_5327 | ABC-type transporter, integral membrane subunit |
| SACT1_5759 | ABC-2 type transporter |
| SACT1_1133 | ABC-type transporter, integral membrane subunit |
| SACT1_1562 | ABC-type transporter, integral membrane subunit |
| SACT1_5518 | major facilitator superfamily MFS_1 |
| SACT1_3430 | ABC-type transporter, integral membrane subunit |
| SACT1_4517 | major facilitator superfamily MFS_1 |
| SACT1_4565 | drug resistance transporter, EmrB/QacA subfamily |
| SACT1_4994 | ABC-type transporter, integral membrane subunit |
| SACT1_7197 | major facilitator superfamily MFS_1 |
| SACT1_5949 | major facilitator superfamily MFS_1 |
| SACT1_6233 | protein of unknown function DUF6 transmembrane |
| SACT1_0936 | ABC-type transporter, integral membrane subunit |
| SACT1_4993 | 2-aminoethylphosphonate ABC transporter, permease protein |
| SACT1_6954 | ABC-type transporter, integral membrane subunit |
| SACT1_1846 | Lysine exporter protein (LYSE/YGGA) |
| SACT1_3429 | ABC-type transporter, integral membrane subunit |
| SACT1_3957 | membrane protein of unknown function |
| SACT1_4612 | ABC-2 type transporter |
| SACT1_1998 | polar amino acid ABC transporter, inner membrane subunit |
| SACT1_2093 | ABC-type transporter, integral membrane subunit |
| SACT1_4420 | ABC-2 type transporter |
| SACT1_6613 | putative ABC transporter permease protein |
| SACT1_2564 | small multidrug resistance protein |
| SACT1_3669 | major facilitator superfamily MFS_1 |
| SACT1_4186 | major facilitator superfamily MFS_1 |
| SACT1_4850 | ABC-type transporter, integral membrane subunit |
| SACT1_0206 | major facilitator superfamily MFS_1 |
| SACT1_5418 | Lysine exporter protein (LYSE/YGGA) |
| SACT1_0548 | ABC-type transporter, integral membrane subunit |
| SACT1_3332 | protein of unknown function DUF6 transmembrane |
| SACT1_3764 | sodium/hydrogen exchanger |
| SACT1_6278 | protein of unknown function DUF6 transmembrane |
| SACT1_4143 | ABC-2 type transporter |
| SACT1_3232 | ABC-type transporter, integral membrane subunit |
| SACT1_0256 | ABC-type transporter, integral membrane subunit |
| SACT1_2898 | major facilitator superfamily MFS_1 |
| SACT1_6510 | protein of unknown function DUF6 transmembrane |
| SACT1_0980 | CrcB-like protein |
| SACT1_1650 | ABC-type transporter, integral membrane subunit |
| SACT1_4658 | acyltransferase 3 |
| SACT1_0306 | protein of unknown function DUF6 transmembrane |
| SACT1_2372 | major facilitator superfamily MFS_1 |
| SACT1_7238 | ABC-type transporter, integral membrane subunit |
| SACT1_0202 | major facilitator superfamily MFS_1 |
| SACT1_0591 | ABC-type transporter, integral membrane subunit |
| SACT1_5369 | protein of unknown function DUF81 |
| SACT1_6227 | C4-dicarboxylate transporter/malic acid transport protein |
| SACT1_6755 | ABC-type transporter, integral membrane subunit |
| SACT1_0978 | Urea transporter |
| SACT1_2418 | ATP synthase subunit a |
| SACT1_5604 | major facilitator superfamily MFS_1 |
| SACT1_4891 | ABC-type transporter, integral membrane subunit |
| SACT1_6507 | protein of unknown function DUF6 transmembrane |
| SACT1_6754 | ABC-type transporter, integral membrane subunit |
| SACT1_0787 | ABC-2 type transporter |
| SACT1_2848 | sodium/hydrogen exchanger |
| SACT1_0636 | ABC-type transporter, integral membrane subunit |
| SACT1_1891 | putative integral membrane protein |
| SACT1_1552 | xanthine permease |
| SACT1_2894 | putative secreted protein |
| SACT1_4508 | sodium:dicarboxylate symporter |
| SACT1_7091 | drug resistance transporter, EmrB/QacA subfamily |
| SACT1_2652 | major facilitator superfamily MFS_1 |
| SACT1_1741 | amino acid permease-associated region |
| SACT1_1838 | ABC-type transporter, integral membrane subunit |
| SACT1_2796 | gluconate transporter |
| SACT1_5220 | sodium/hydrogen exchanger |
| SACT1_6991 | ABC-type transporter, integral membrane subunit |
| SACT1_3273 | protein of unknown function DUF894 DitE |
| SACT1_7089 | ABC-type transporter, integral membrane subunit |
| SACT1_7280 | major facilitator superfamily MFS_1 |
| SACT1_3467 | major facilitator superfamily MFS_1 |
| SACT1_1304 | ABC-type transporter, integral membrane subunit |
| SACT1_6032 | protein of unknown function DUF81 |
| SACT1_4312 | major facilitator superfamily MFS_1 |
| SACT1_0876 | ABC-type transporter, integral membrane subunit |
| SACT1_6123 | citrate/H+ symporter, CitMHS family |
| SACT1_4359 | Cl- channel voltage-gated family protein |
| SACT1_7325 | branched-chain amino acid transport |
| SACT1_1160 | protein of unknown function DUF140 |
| SACT1_2265 | Arsenical pump membrane protein |
| SACT1_3512 | ABC-2 type transporter |
| SACT1_1018 | major facilitator superfamily MFS_1 |
| SACT1_3415 | Xanthine/uracil/vitamin C permease |
| SACT1_5214 | BioY protein |
| SACT1_3656 | small multidrug resistance protein |
| SACT1_3895 | SpdD2 protein |
| SACT1_4929 | ABC-type transporter, integral membrane subunit |
| SACT1_3029 | major facilitator superfamily MFS_1 |
| SACT1_6312 | ABC-type transporter, integral membrane subunit |
| SACT1_0919 | L-lactate transport |
| SACT1_4356 | ABC-2 type transporter |
| SACT1_0532 | ABC-type transporter, integral membrane subunit |
| SACT1_6693 | secretion protein snm4 |
| SACT1_0967 | ABC-type transporter, integral membrane subunit |
| SACT1_6496 | major facilitator superfamily MFS_1 |
| SACT1_6983 | major facilitator superfamily permease |
| SACT1_0917 | NADH-ubiquinone/plastoquinone oxidoreductase chain 3 |
| SACT1_6887 | major facilitator superfamily MFS_1 |
| SACT1_2835 | major facilitator superfamily MFS_1 |
| SACT1_5544 | drug resistance transporter, Bcr/CflA subfamily |
| SACT1_5591 | ABC-type transporter, integral membrane subunit |
| SACT1_1201 | ABC-type transporter, integral membrane subunit |
| SACT1_2404 | ABC-2 type transporter |
| SACT1_0870 | protein of unknown function DUF803 |
| SACT1_6933 | ABC-type transporter, integral membrane subunit |
| SACT1_1776 | ABC-type transporter, integral membrane subunit |
| SACT1_3213 | major facilitator superfamily MFS_1 |
| SACT1_4210 | phosphate ABC transporter, inner membrane subunit PstC |
| SACT1_5398 | protein of unknown function DUF81 |
| SACT1_0914 | NADH-ubiquinone/plastoquinone oxidoreductase chain 6 |
| SACT1_3261 | major facilitator superfamily MFS_1 |
| SACT1_6932 | ABC-type transporter, integral membrane subunit |
| SACT1_0669 | major facilitator superfamily MFS_1 |
| SACT1_4255 | ABC-2 type transporter |
| SACT1_4541 | major facilitator superfamily MFS_1 |
| SACT1_4638 | major facilitator superfamily MFS_1 |
| SACT1_0913 | NADH-ubiquinone oxidoreductase chain 4L |
| SACT1_4443 | protein of unknown function DUF81 |
| SACT1_5396 | Xanthine/uracil/vitamin C permease |
| SACT1_0912 | NADH dehydrogenase (quinone) |
| SACT1_2924 | Lysine exporter protein (LYSE/YGGA) |
| SACT1_5922 | putative integral membrane protein |
| SACT1_1243 | Bile acid:sodium symporter |
| SACT1_6967 | ABC-type transporter, integral membrane subunit |
| SACT1_0911 | proton-translocating NADH-quinone oxidoreductase, chain M |
| SACT1_3931 | putative ABC transporter permease protein |
| SACT1_2820 | ABC-2 type transporter |
| SACT1_3298 | putative integral membrane transport protein |
| SACT1_4871 | major facilitator superfamily MFS_1 |
| SACT1_5873 | major facilitator superfamily MFS_1 |
| SACT1_6636 | putative integral membrane protein |
| SACT1_0905 | AbgT transporter |
| SACT1_5532 | 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase |
| SACT1_0910 | proton-translocating NADH-quinone oxidoreductase, chain N |
| SACT1_2115 | ABC-type transporter, integral membrane subunit |
| SACT1_4635 | protein of unknown function DUF6 transmembrane |
| SACT1_5777 | major facilitator superfamily MFS_1 |
| SACT1_3979 | major facilitator superfamily MFS_1 |
| SACT1_5536 | major facilitator superfamily MFS_1 |
| SACT1_6782 | major facilitator superfamily MFS_1 |
| SACT1_0616 | virulence factor MVIN family protein |
| SACT1_4869 | ABC-type transporter, integral membrane subunit |
| SACT1_1581 | putative integral membrane protein |
| SACT1_4585 | major facilitator superfamily MFS_1 |
| SACT1_6536 | small multidrug resistance protein |
| SACT1_4024 | cell cycle protein |
| SACT1_5296 | major facilitator superfamily MFS_1 |
| SACT1_1865 | major facilitator superfamily MFS_1 |
| SACT1_4868 | ABC-type transporter, integral membrane subunit |
| SACT1_0955 | protein of unknown function DUF803 |
| SACT1_4296 | major facilitator superfamily MFS_1 |
| SACT1_5104 | major facilitator superfamily MFS_1 |
| SACT1_0519 | Bile acid:sodium symporter |
| SACT1_2394 | putative ABC transporter permease protein |
| SACT1_0661 | major facilitator superfamily MFS_1 |
| SACT1_2062 | major facilitator superfamily MFS_1 |
| SACT1_4295 | ABC-type transporter, integral membrane subunit |
| SACT1_6828 | peptidase M48 Ste24p |
| SACT1_3446 | major facilitator superfamily MFS_1 |
| SACT1_6631 | Lysine exporter protein (LYSE/YGGA) |
| SACT1_1048 | ABC-type transporter, integral membrane subunit |
| SACT1_1528 | protein of unknown function DUF6 transmembrane |
| SACT1_2016 | branched-chain amino acid transport |
| SACT1_6154 | gluconate transporter |
| SACT1_5051 | major facilitator superfamily MFS_1 |
| SACT1_5531 | protein of unknown function DUF81 |
| SACT1_6480 | protein of unknown function DUF1290 |
| SACT1_0373 | ABC-type transporter, integral membrane subunit |
| SACT1_2392 | putative ABC transporter permease protein |
| SACT1_2724 | protein of unknown function DUF107 |
| SACT1_7257 | protein of unknown function UPF0118 |
| SACT1_2772 | putative integral membrane protein |
| SACT1_3201 | NAD(P)H-quinone oxidoreductase subunit 4L |
| SACT1_7066 | ABC-type transporter, integral membrane subunit |
Some of these genes are labeled "protein of unknown function," but I think we can predict with high confidence, based on what we know about these proteins (namely, that they're hydrophobic) that the gene products in question involve membrane-associated functions.
Bioinformatics geeks, leave a comment below.
