Generalizing the isothermal efficiency by using Gaussian distributions.

Unlike the Carnot heat engine efficiency published in 1824, an isothermal efficiency derived from thermodynamics and information theory can be applied to biological systems. The original approach by Pierce and Cutler in 1959 to derive the isothermal efficiency equation came from Shannon's channel capacity of 1949 and from Felker's 1952 determination of the minimum energy dissipation needed to gain a bit. In 1991 and 2010 Schneider showed how the isothermal efficiency equation can be applied to molecular machines and that this can be used to explain why several molecular machines are 70% efficient. Surprisingly, some macroscopic biological systems, such as whole ecosystems, are also 70% efficient but it is hard to see how this could be explained by a thermodynamic and molecular theory. The thesis of this paper is that the isothermal efficiency can be derived without using thermodynamics by starting from a set of independent Gaussian distributions. This novel derivation generalizes the isothermal efficiency equation for use at all levels of biology, from molecules to ecosystems.

Restriction enzymes use a 24 dimensional coding space to recognize 6 base long DNA sequences.

Restriction enzymes recognize and bind to specific sequences on invading bacteriophage DNA. Like a key in a lock, these proteins require many contacts to specify the correct DNA sequence. Using information theory we develop an equation that defines the number of independent contacts, which is the dimensionality of the binding. We show that EcoRI, which binds to the sequence GAATTC, functions in 24 dimensions. Information theory represents messages as spheres in high dimensional spaces. Better sphere packing leads to better communications systems. The densest known packing of hyperspheres occurs on the Leech lattice in 24 dimensions. We suggest that the single protein EcoRI molecule employs a Leech lattice in its operation. Optimizing density of sphere packing explains why 6 base restriction enzymes are so common.

Elements in the λ immunity region regulate phage development: beyond the 'Genetic Switch'.

Genetic elements in the bacteriophage λ immunity region contribute to stable maintenance and synchronous induction of the integrated Escherichia coli prophage. There is a bistable switch between lysogenic and lytic growth that is orchestrated by the CI and Cro repressors acting on the lytic (PL and PR ) and lysogenic (PRM ) promoters, referred to as the Genetic Switch. Other less well-characterized elements in the phage immunity region include the PLIT promoter and the immunity terminator, TIMM . The PLIT promoter is repressed by the bacterial LexA protein in λ lysogens. LexA repressor, like the λ CI repressor, is inactivated during the SOS response to DNA damage, and this regulation ensures that the PLIT promoter and the lytic PL and PR promoters are synchronously activated. Proper RexA and RexB protein levels are critical for the switch from lysogeny to lytic growth. Mutation of PLIT reduces RexB levels relative to RexA, compromising cellular energetics and causing a 10-fold reduction in lytic phage yield. The RexA and RexB proteins interact with themselves and each other in a bacterial two-hybrid system. We also find that the transcription terminator, TIMM , is a Rho-independent, intrinsic terminator. Inactivation of TIMM has minimal effect on λ lysogenization or prophage induction.

Density of σ70 promoter-like sites in the intergenic regions dictates the redistribution of RNA polymerase during osmotic stress in Escherichia coli.

RNA polymerase (RNAP), the transcription machinery, shows dynamic binding across the genomic DNA under different growth conditions. The genomic features that selectively redistribute the limited RNAP molecules to dictate genome-wide transcription in response to environmental cues remain largely unknown. We chose the bacterial osmotic stress response model to determine genomic features that direct genome-wide redistribution of RNAP during the stress. Genomic mapping of RNAP and transcriptome profiles corresponding to the different temporal states after salt shock were determined. We found rapid redistribution of RNAP across the genome, primarily at σ70 promoters. Three subsets of genes exhibiting differential salt sensitivities were identified. Sequence analysis using an information-theory based σ70 model indicates that the intergenic regions of salt-responsive genes are enriched with a higher density of σ70 promoter-like sites than those of salt-sensitive genes. In addition, the density of promoter-like sites has a positive linear correlation with RNAP binding at different salt concentrations. The RNAP binding contributed by the non-initiating promoter-like sites is important for gene transcription at high salt concentration. Our study demonstrates that hyperdensity of σ70 promoter-like sites in the intergenic regions of salt-responsive genes drives the RNAP redistribution for reprograming the transcriptome to counter osmotic stress.

An Evolutionary/Biochemical Connection between Promoter- and Primer-Dependent Polymerases Revealed by Systematic Evolution of Ligands by Exponential Enrichment.

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified systematic evolution of ligands by exponential enrichment (SELEX) approach. Two Taq-specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. TaqI contained 8 nucleotides (5'-CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCRs. Similarly, exonuclease- Klenow polymerase also selected a high-affinity primer that contained a related core promoter sequence from phage T7 RNAP (5'-ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity, suggesting that binding was highly sequence specific. The results are discussed in the context of possible effects on multiprimer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs.IMPORTANCE This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function or be a consequence of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered, and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promoters. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology.

Genome-Wide Transcriptional Regulation and Chromosome Structural Arrangement by GalR in E. coli.

The regulatory protein, GalR, is known for controlling transcription of genes related to D-galactose metabolism in Escherichia coli. Here, using a combination of experimental and bioinformatic approaches, we identify novel GalR binding sites upstream of several genes whose function is not directly related to D-galactose metabolism. Moreover, we do not observe regulation of these genes by GalR under standard growth conditions. Thus, our data indicate a broader regulatory role for GalR, and suggest that regulation by GalR is modulated by other factors. Surprisingly, we detect regulation of 158 transcripts by GalR, with few regulated genes being associated with a nearby GalR binding site. Based on our earlier observation of long-range interactions between distally bound GalR dimers, we propose that GalR indirectly regulates the transcription of many genes by inducing large-scale restructuring of the chromosome.

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

Promoter variants in the MSMB gene associated with prostate cancer regulate MSMB/NCOA4 fusion transcripts.

Beta-microseminoprotein (MSP)/MSMB is an immunoglobulin superfamily protein synthesized by prostate epithelial cells and secreted into seminal plasma. Variants in the promoter of the MSMB gene have been associated with the risk of prostate cancer (PCa) in several independent genome-wide association studies. Both MSMB and an adjacent gene, NCOA4, are subjected to transcriptional control via androgen response elements. The gene product of NCOA4 interacts directly with the androgen receptor as a co-activator to enhance AR transcriptional activity. Here, we provide evidence for the expression of full-length MSMB-NCOA4 fusion transcripts regulated by the MSMB promoter. The predominant MSMB-NCOA4 transcript arises by fusion of the 5'UTR and exons 1-2 of the MSMB pre-mRNA, with exons 2-10 of the NCOA4 pre-mRNA, producing a stable fusion protein, comprising the essential domains of NCOA4. Analysis of the splice sites of this transcript shows an unusually strong splice acceptor at NCOA4 exon 2 and the presence of Alu repeats flanking the exons potentially involved in the splicing event. Transfection experiments using deletion clones of the promoter coupled with luciferase reporter assays define a core MSMB promoter element located between -27 and -236 of the gene, and a negative regulatory element immediately upstream of the start codon. Computational network analysis reveals that the MSMB gene is functionally connected to NCOA4 and the androgen receptor signaling pathway. The data provide an example of how GWAS-associated variants may have multiple genetic and epigenetic effects.

An unusual feature associated with LEE1 P1 promoters in enteropathogenic Escherichia coli (EPEC).

Transcription start points in bacteria are influenced by the nature of the RNA polymerase·promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing σ70, it is presumed that specific sequence in one or more of the -10, extended -10 and -35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF.

70% efficiency of bistate molecular machines explained by information theory, high dimensional geometry and evolutionary convergence.

The relationship between information and energy is key to understanding biological systems. We can display the information in DNA sequences specifically bound by proteins by using sequence logos, and we can measure the corresponding binding energy. These can be compared by noting that one of the forms of the second law of thermodynamics defines the minimum energy dissipation required to gain one bit of information. Under the isothermal conditions that molecular machines function this is Emin = Kb T ln 2 joules per bit (kB is Boltzmann's constant and T is the absolute temperature). Then an efficiency of binding can be computed by dividing the information in a logo by the free energy of binding after it has been converted to bits. The isothermal efficiencies of not only genetic control systems, but also visual pigments are near 70%. From information and coding theory, the theoretical efficiency limit for bistate molecular machines is ln 2=0.6931. Evolutionary convergence to maximum efficiency is limited by the constraint that molecular states must be distinct from each other. The result indicates that natural molecular machines operate close to their information processing maximum (the channel capacity), and implies that nanotechnology can attain this goal.

A brief review of molecular information theory.

The idea that we could build molecular communications systems can be advanced by investigating how actual molecules from living organisms function. Information theory provides tools for such an investigation. This review describes how we can compute the average information in the DNA binding sites of any genetic control protein and how this can be extended to analyze its individual sites. A formula equivalent to Claude Shannon's channel capacity can be applied to molecular systems and used to compute the efficiency of protein binding. This efficiency is often 70% and a brief explanation for that is given. The results imply that biological systems have evolved to function at channel capacity, which means that we should be able to build molecular communications that are just as robust as our macroscopic ones.

Discovery of novel tumor suppressor p53 response elements using information theory.

An accurate method for locating genes under tumor suppressor p53 control that is based on a well-established mathematical theory and built using naturally occurring, experimentally proven p53 sites is essential in understanding the complete p53 network. We used a molecular information theory approach to create a flexible model for p53 binding. By searching around transcription start sites in human chromosomes 1 and 2, we predicted 16 novel p53 binding sites and experimentally demonstrated that 15 of the 16 (94%) sites were bound by p53. Some were also bound by the related proteins p63 and p73. Thirteen of the adjacent genes were controlled by at least one of the proteins. Eleven of the 16 sites (69%) had not been identified previously. This molecular information theory approach can be extended to any genetic system to predict new sites for DNA-binding proteins.

Xeroderma pigmentosum-variant patients from America, Europe, and Asia.

Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.

Small membrane proteins found by comparative genomics and ribosome binding site models.

The correct annotation of genes encoding the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short open reading frames have been confirmed to correspond to synthesized proteins. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16-50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated genes by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. We summarize the properties of these small proteins; remarkably more than half of the proteins are predicted to be single-transmembrane proteins, nine of which we show co-fractionate with cell membranes.

Discovery of Fur binding site clusters in Escherichia coli by information theory models.

Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints ( approximately 83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur-DNA recognition mechanism may be conserved for even distantly related bacteria.

Correlation between binding rate constants and individual information of E. coli Fis binding sites.

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.

Anatomy of Escherichia coli sigma70 promoters.

Information theory was used to build a promoter model that accounts for the -10, the -35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base -7, rather than -11, of the -10 may be flipping to initiate transcription. We found that the sequence conservation of sigma70 binding sites is 6.5 +/- 0.1 bits. Some promoters lack a -35 region, but have a 6.7 +/- 0.2 bit extended -10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended -10 binding and the -35 suggest that the flexible bipartite sigma factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5' mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the -35, so bending allows distal activators to reach the polymerase.

CorreLogo: an online server for 3D sequence logos of RNA and DNA alignments.

We present an online server that generates a 3D representation of properties of user-submitted RNA or DNA alignments. The visualized properties are information of single alignment columns, mutual information of two alignment positions as well as the position-specific fraction of gaps. The nucleotide composition of both single columns and column pairs is visualized with the help of color-coded 3D bars labeled with letters. The server generates both VRML and JVX output that can be viewed with a VRML viewer or the JavaView applet, respectively. We show that combining these different features of an alignment into one 3D representation is helpful in identifying correlations between bases and potential RNA and DNA base pairs. Significant known correlations between the tRNA 3' anticodon cardinal nucleotide and the extended anticodon were observed, as were correlations within the amino acid acceptor stem and between the cardinal nucleotide and the acceptor stem. The online server can be accessed using the URL http://correlogo.abcc.ncifcrf.gov.

Comparative analysis of tandem T7-like promoter containing regions in enterobacterial genomes reveals a novel group of genetic islands.

Based on molecular information theory, 10 T7-like promoter models were built for the T7 group of phages and used to scan their host genomes and closely related genomes. 38 genomes were scanned and 12 clusters of tandem promoters were identified in nine enteropathogens. Comparative analysis of these tandem promoter-bearing regions reveals that they are similar to each other, forming prophage-like islands of 4-13 kb. Each island appears to contain two or three tandem T7-like promoters within a stretch of 150-620 bases, but there are no corresponding RNA polymerase (RNAP) genes. The promoters would transcribe two to five putative phage-related proteins, but none of these resemble known phage structural proteins. An integrase belonging to the Int family of site-specific recombinases is encoded upstream of the tandem promoters. A direct repeat of 17-24 bases was found on the ends of all 12 islands. Comparative analysis of the islands shows that these islands appear to have recombined with each other. These results suggest that the islands could encode a group of satellite phages. Activation and function of the islands may depend on transcription by a T7-like RNAP after infection by a T7-like phage or foreign DNA that encodes a T7-like RNAP.