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| Mycoplasma genitalium |
Recently, I looked at the genome of M. genitalium from the point of view of latent nucleic-acid secondary structure. (Secondary structure refers to the ability of a single strand of DNA or RNA to base-pair with itself.) I probed each gene with a script designed to detect intragenic self-complementing regions of length eleven (so-called 11-mers), the idea being that complementary runs of bases shorter than that might occur at a high rate by chance. Given M. genitalium's base composition stats (adenine being most prevalent, at 36.24% of all bases in protein-coding message regions, thymine being next-most-abundant, at 32.21% of protein-coding bases), the most likely 11-mer, 'AAAAAAAAAAA', could be expected to occur by chance once every 70,672 bases. In actuality, that particular 11-mer doesn't occur in the M. genitalium genome, but if it did we could expect around 8 occurrences of it in a genome of 528,500 base pairs. Instead, what we actually find are 749 occurrences of complementary 11-mers in 283 genes.
The procedure used to check for 11-mers is as follows (in pseudocode):
for ( i = 0; i < genes.length; i++ ) // for each gene for ( k = 0; k < genes[i].length - 11; k++ ) // for all bases sequence = genes[i].substring( k, k + 11 ) // get 11 bases complement = getReverseComplement( sequence ) if ( genes[i].match( complement ) ) // a match exists if ( matchNotWithinMask( mask ) ) // not inside a previous match if ( matchNotInsideSequence( sequence ) ) // not inside sequence matches++ // count it as a hit updateMask( ) // update mask
Note that with even-length sequences it would be important to guard against self-matching sequences, since a sequence like AGCT is its own reverse-complement. With length-11 sequences this is not an issue. Nevertheless, it's possible for successive matches to overlap, and I wrote a check to guard against that. That's what matchNotWithinMask( mask ) and updateMask() are all about.
It should go without saying, but I'll say it anyway: 749 complementing 11-mers in 283 genes is a very substantial (and quite unexpected) amount of internal complementarity. The question is: What's the purpose of all that (putative) secondary structure?
One possibility (which I've talked about in a previous post) is that the secondary structure allows for thermostatic control of gene expression. RNA thermometers are a well established phenomenon and it could be that M. genitalium needs sensitive control over thermal expression of certain genes.
Another possibility is that many genes incorporate magnesium-, manganese-, or calcium-sensitive riboswitches. RNA is a potent chelator of metal ions, particularly doubly-charged ions like Mg+2, which is smaller than sodium or potassium (with twice the charge) and thus has unusually high charge density. It's possible that under conditions of osmotic stress (with high influx of water and low ion concentrations) certain mRNAs relax or uncoil and thereby become translatable. If this is true (if certain genes are under osmotic control), we might expect to find that many M. genitalium genes with high secondary structure potential are membrane-targeted genes tasked with managing the import and export of various things. And that's indeed what we find.
Further below, I present a table with the top 100 M. genitalium genes containing the most putative secondary structure based on the 11-mer probe outlined above. The table shows the gene name, gene product or function (where known), gene size in base pairs, and the number of complementing 11-mers in the gene. The genes tend to fall into only a few categories. About a third of the genes (34 out of 100) are DNA- or RNA-binding genes. A dozen genes are transporters or permeases; another 12 fall in the category of "other membrane-associated genes" (these are marked with asterisks); and ten encode lipoproteins. Sixteen are "hypothetical proteins" (which should probably be reannotated as proteins of unknown function, since we can be fairly sure the genes are expressed).
It's interesting that so many genes with (putative) secondary-structure potential are nucleic acid processing genes. Among genes in this category are ten genes that either acylate or modify transfer RNAs. What's interesting about the latter group is that most involve tRNAs for non-polar amino acids (alanine, leucine, isoleucine, valine, phenylalanine, and methionine). The reason this is interesting is that non-polar amino acids are extensively used in membrane-associated proteins. Thus we have a situation, possibly, in which osmo-switches (osmotically sensitive mRNAs) control the expression of tRNA synthetases for amino acids used in membrane proteins.
It is quite possible that some of the (few) metabolic genes listed in the table are membrane-associated. This is likely true for phosphomannomutase, UDP-galactopyranose mutase, and glycerophosphoryl diester phosphodiesterase, for example. Also interesting is that 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from Thermoplasma has been shown to be manganese-stimulated. Riboswitch modulation of such an enzyme would not be unexpected.
Altogether, 34 to 37 out of 100 genes listed in the table are membrane-associated, and another 7 are tRNA synthetases involving non-polar amino acids (heavily used in membrane proteins), tending to support the hypothesis that M. genitalium uses secondary structure of mRNA (and/or ssDNA) to modulate gene expression in osmotically sensitive manner.
Table 1. Genes with high secondary structure potential in M. genitalium. Genes marked with asterisks are membrane-associated genes other than transporters or permeases. The final column shows the number of complementary 11-mer pairs found in the gene.
| Gene | Product |
Size
(bp)
|
11-mers
|
| MG_468 | ABC transporter, permease protein |
5353
|
22
|
| MG_064 | ABC transporter, permease protein, putative |
3997
|
17
|
| MG_218 | HMW2 cytadherence accessory protein * |
5419
|
16
|
| MG_386 | P200 protein |
4852
|
15
|
| MG_414 | conserved hypothetical protein |
3112
|
13
|
| MG_075 | 116 kDa surface antigen * |
3076
|
12
|
| MG_422 | conserved hypothetical protein |
2509
|
12
|
| MG_244 | UvrD/REP helicase |
2113
|
11
|
| MG_191 | MgPa adhesin * |
4336
|
11
|
| MG_292 | alanyl-tRNA synthetase |
2704
|
10
|
| MG_018 | helicase SNF2 family, putative |
3097
|
10
|
| MG_298 | chromosome segregation protein SMC |
2950
|
10
|
| MG_345 | isoleucyl-tRNA synthetase |
2689
|
9
|
| MG_338 | lipoprotein, putative |
3814
|
9
|
| MG_307 | lipoprotein, putative |
3535
|
8
|
| MG_031 | DNA polymerase III, alpha subunit, Gram-positive type |
4357
|
8
|
| MG_080 | oligopeptide ABC transporter, ATP-binding protein |
2548
|
8
|
| MG_321 | lipoprotein, putative |
2806
|
7
|
| MG_340 | DNA-directed RNA polymerase, beta' subunit |
3880
|
7
|
| MG_069 | PTS system, glucose-specific IIABC component * |
2728
|
7
|
| MG_390 | ABC transporter, ATP-binding/permease protein |
1984
|
7
|
| MG_525 | conserved hypothetical protein |
1996
|
7
|
| MG_226 | amino acid-polyamine-organocation (APC) permease family protein |
1480
|
6
|
| MG_378 | arginyl-tRNA synthetase |
1615
|
6
|
| MG_192 | P110 protein |
3163
|
6
|
| MG_312 | HMW1 cytadherence accessory protein * |
3421
|
6
|
| MG_328 | conserved hypothetical protein |
2272
|
6
|
| MG_341 | DNA-directed RNA polymerase, beta subunit |
4174
|
6
|
| MG_291 | phosphonate ABC transporter, permease protein (P69), putative |
1633
|
5
|
| MG_001 | DNA polymerase III, beta subunit |
1144
|
5
|
| MG_411 | phosphate ABC transporter, permease protein PstA |
1966
|
5
|
| MG_136 | lysyl-tRNA synthetase |
1474
|
5
|
| MG_336 | aminotransferase, class V |
1228
|
5
|
| MG_261 | DNA polymerase III, alpha subunit |
2626
|
5
|
| MG_430 | 2,3-bisphosphoglycerate-independent phosphoglycerate mutase |
1525
|
5
|
| MG_053 | phosphoglucomutase/phosphomannomutase, putative |
1654
|
5
|
| MG_195 | phenylalanyl-tRNA synthetase, beta subunit |
2422
|
5
|
| MG_260 | lipoprotein, putative |
2299
|
5
|
| MG_277 | membrane protein, putative * |
2914
|
5
|
| MG_366 | conserved hypothetical protein |
2005
|
5
|
| MG_250 | DNA primase |
1825
|
4
|
| MG_447 | membrane protein, putative * |
1645
|
4
|
| MG_254 | DNA ligase, NAD-dependent |
1981
|
4
|
| MG_003 | DNA gyrase, B subunit |
1954
|
4
|
| MG_397 | conserved hypothetical protein |
1702
|
4
|
| MG_096 | conserved hypothetical protein |
1954
|
4
|
| MG_334 | valyl-tRNA synthetase |
2515
|
4
|
| MG_141 | transcription termination factor NusA |
1597
|
4
|
| MG_241 | conserved hypothetical protein |
1864
|
4
|
| MG_278 | GTP pyrophosphokinase |
2164
|
4
|
| MG_012 | alpha-L-glutamate ligases, RimK family, putative |
865
|
4
|
| MG_123 | conserved hypothetical protein |
1417
|
4
|
| MG_266 | leucyl-tRNA synthetase |
2380
|
4
|
| MG_119 | ABC transporter, ATP-binding protein |
1696
|
4
|
| MG_068 | lipoprotein, putative |
1426
|
3
|
| MG_047 | S-adenosylmethionine synthetase |
1153
|
3
|
| MG_456 | conserved hypothetical protein |
1006
|
3
|
| MG_122 | DNA topoisomerase I |
2131
|
3
|
| MG_421 | excinuclease ABC, A subunit |
2866
|
3
|
| MG_223 | conserved hypothetical protein |
1237
|
3
|
| MG_375 | threonyl-tRNA synthetase |
1696
|
3
|
| MG_364 | expressed protein of unknown function |
676
|
3
|
| MG_185 | lipoprotein, putative |
2107
|
3
|
| MG_423 | conserved hypothetical protein |
1687
|
3
|
| MG_067 | lipoprotein, putative |
1552
|
3
|
| MG_008 | tRNA modification GTPase TrmE |
1330
|
3
|
| MG_306 | membrane protein, putative * |
1183
|
3
|
| MG_242 | expressed protein of unknown function |
1894
|
3
|
| MG_040 | lipoprotein, putative |
1777
|
3
|
| MG_281 | conserved hypothetical protein |
1672
|
3
|
| MG_259 | modification methylase, HemK family |
1372
|
3
|
| MG_204 | DNA topoisomerase IV, A subunit |
2347
|
3
|
| MG_216 | pyruvate kinase |
1528
|
3
|
| MG_229 | ribonucleoside-diphosphate reductase, beta chain |
1024
|
3
|
| MG_187 | ABC transporter, ATP-binding protein |
1759
|
3
|
| MG_094 | replicative DNA helicase |
1408
|
3
|
| MG_184 | adenine-specific DNA modification methylase |
955
|
3
|
| MG_303 | metal ion ABC transporter, ATP-binding protein, putative |
1075
|
3
|
| MG_045 | spermidine/putrescine ABC transporter, spermidine/putrescine binding protein, putative |
1453
|
3
|
| MG_051 | pyrimidine-nucleoside phosphorylase |
1267
|
3
|
| MG_004 | DNA gyrase, A subunit |
2512
|
3
|
| MG_385 | glycerophosphoryl diester phosphodiesterase family protein * |
712
|
3
|
| MG_360 | ImpB/MucB/SamB family protein |
1237
|
3
|
| MG_457 | ATP-dependent metalloprotease FtsH * |
2110
|
3
|
| MG_065 | ABC transporter, ATP-binding protein |
1402
|
3
|
| MG_419 | DNA polymerase III, subunit gamma and tau |
1795
|
3
|
| MG_295 | tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase |
1105
|
3
|
| MG_072 | preprotein translocase, SecA subunit * |
2422
|
3
|
| MG_464 | membrane protein, putative * |
1159
|
3
|
| MG_089 | translation elongation factor G |
2068
|
2
|
| MG_439 | lipoprotein, putative |
820
|
2
|
| MG_309 | lipoprotein, putative |
3679
|
2
|
| MG_032 | conserved hypothetical protein |
2002
|
2
|
| MG_194 | phenylalanyl-tRNA synthetase, alpha subunit |
1027
|
2
|
| MG_137 | UDP-galactopyranose mutase |
1216
|
2
|
| MG_203 | DNA topoisomerase IV, B subunit |
1903
|
2
|
| MG_021 | methionyl-tRNA synthetase |
1540
|
2
|
| MG_029 | DJ-1/PfpI family protein |
562
|
2
|
| MG_255 | conserved hypothetical protein |
1099
|
2
|
| MG_314 | conserved hypothetical protein |
1333
|
2
|
