Monthly Archives: April 2012

On Ribosomal Pausing

A new paper in Nature, describes how Shine-Dalgarno-like features in protein coding sequences, leads to bacterial ribosomes pausing during translation. Selection against ribosomal pausing leads to biases in codon usage and coding sequence evolution. Translational pausing represents a new level of regulatory control of gene expression.

Translation, the process by which the nucleotide sequence of mRNA transcripts is decoded and converted into amino acid sequence during protein synthesis, is carried out by ribosomes. Within the ribosome, transfer RNA molecules recognise specific trinucleotide codons on the mRNA, and add their cognate amino acids to nascent protein chains. In bacteria and archaea, ribosomes often recognise the translation start site with the help of a sequence 8 to 12 nucleotides upstream of it – the Shine-Dalgarno sequence (SD). It’s been known since the 1980s that different mRNAs are translated at different rates. The main reason for these differences was thought to be the concentration of rarer varieties of tRNA limiting the rate at which some transcripts could be decoded.

Li et al. have used a new technique, ribosome profiling, that maps ribosome occupancy along mRNAs. This has yielded high-resolution views of local translation rates on the entire protein coding transcriptome of E. coli and Bacillus subtilis.  Briefly put, mRNA fragments that have been protected from nuclease digestion by ribosomal binding, are ‘deep sequenced’. The concentration of these ribosome footprints equates to the ribosome occupancy throughout the transcriptome. The local translation rate is inversely related to ribosome occupancy.

Using this technique, Li et al. found many sites where ribosomal density is ten fold or more than the mean. They sought to correlate these translational pauses with specific codons. However, there was little link between occupancy of specifc codons and the abundance of their corresponding tRNAs. Therefore, the concentration of rare tRNAs is not responsible for much translational pausing under normal growth conditions.

To try to find sequence features that were linked to ribosomal pausing, the researchers then tried to correlate any trinucleotide sequences (independently of reading frame) with ribosome occupancy. They found that 6 different trinucleotide sequences, with features resembling Shine-Dalgarno sequences, did correlate with the position of paused ribosomes. This correlation was not found in the yeast, Saccharomyces cerevisiae; in agreement with eukaryotic ribosomes not using SD- anti-SD interactions.

Li et al. went on to show definitively that internal SD-like sequences are linked to translational pausing, by introducing a mutation into one such site and showing that ribosome occupancy was reduced. They also showed that peaks of ribosome occupancy, were caused by translational pausing, rather than attempted internal translational initiation.

As translational pausing limits the amount of free ribosomes, widespread internal SD-like sequences would limit the rate of protein synthesis, and hence the potential bacterial growth rate. In line with this, SD-like sequences in protein coding genes are disfavoured. Selection pressure against SD-like sequences is therefore a major factor in determining codon usage, and more especially the usage of codon pairs (SD sequences are 6/7 nt long).

Interestingly, the authors found that patterns of ribosome occupancy were conserved between orthologous genes in E. coli and B. subtilis. This reflects two different factors; firstly, coding is obviously constrained by protein’s functionality, but secondly it’s suggestive of translational pausing being exploited for functional purposes. Li et al. suggest a number of different ways in which ribosomal pausing can regulate gene expression. It’s known that internal SD-like sequences can promote regulated shifting of reading frame. Ribosome pausing may also modulate folding of nascent protein chains. Lastly, as transcription and translation are closely coupled in bacteria, ribosome occupancy may inhibit the formation of stem-loop structures that prevent transcriptional termination. It will be exciting to work out the extents to which these potential regulatory systems are active. Eukaryotic ribosomes do not use recognition of SD sequences, instead using the 5’ mRNA cap and the less well defined Kozak sequence for translational initiation. Does ribosome pausing occur in eukaryotes? and does it have functional significance?

Li, G., Oh, E., & Weissman, J. (2012). The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria Nature, 484 (7395), 538-541 DOI: 10.1038/nature10965

Prions, more than just brain rot.

Prions, self-replicating proteins, the causative agents underlying BSE and CJD, have potentially important roles in evolution and memory formation.

Here in the UK, we don’t need reminding about the horrific consequences of transmissable spongiform encephalopathies. Over one hundred and fifty britons have died of variant Creutzfeld-Jacob disease, and the images of cattle suffering the effects of BSE (and ministers feeding their daughters burgers) are still fresh in the mind. Part of the difficulty felt by scientists and government in handling the BSE crisis were due to these diseases being caused by a novel form of pathogenic entity, prions. Rather than encoding information facilitating their replication and transmission in a DNA or RNA genome like viruses and other pathogens, prions are proteins capable of self-replication. More specifically they are an aberrant conformation of a protein, capable of seeding the misfolding of the native form. In the case of the spongiform encephalopathies, the native ‘prion protein’ (a component of neuronal membranes, still of unknown function) is converted into a tangled form that is resistant to enzymatic degradation. This prion form is therefore capable of transmission through digestive systems that would normally degrade proteins. In the brain, the prions form toxic aggregations, causing neurodegeneration and death.

The idea that self-replicating proteins could act as elements of inheritance was revolutionary at the time, but prions are now being found to exist in many other contexts, and rather than acting as pathogens, their potential functions are yielding exciting insights into evolution and brain function.

In yeast, at least nine different proteins have been shown to form prions, and eighteen more contain prion-forming domains. These are often important proteins involved in the control of cellular regulation. The best characterised yeast prion, [ PSI+], is a form of a factor responsible for the termination of translation (the process of converting the sequence of messenger RNA transcripts into the amino acid sequence of protein), Sup35. [ PSI+] titrates normal Sup35 protein, lessening its level, and leading to an increase in translational read-through. This read-through effectively uncovers cryptic genetic variation. Genetic sequence that is not normally encoding protein sequence will be under less stringent evolutionary selection pressure than coding sequence. If this sequence is suddenly translated into protein in [ PSI+] cells it may, in a minority of cases, be beneficial for cell’s adaptation to their environment. Protein folding is mediated by ‘chaperone’ proteins, which are also closely involved in the response to environmental stresses. Hence, prion formation, a case of protein ‘mis-folding’, is more likely to occur during times of stress. Prions can therefore act as switches responsible for the sudden appearance of complex traits in response to environmental conditions.  Although these possible ‘evolvability’ roles for prions in yeast are controversial, it appears that the prion-forming domains responsible for this capacity, have been conserved for long periods of evolutionary time, and do not generally have other major functions.  Recently, [ PSI+] and another yeast prion have been shown to exist in wild yeast strains, strengthening the argument that they are not simply diseases or artifacts of laboratory culture.

Proteins that contain prion-forming domains are present in many branches of the tree of life. A particularly exciting example, found in the sea slug Aplysia, is an RNA-binding protein called CPEB. This protein is responsible for regulating the activation of the translation of mRNA transcripts in neuronal synapses in response to neurotransmitters, such as serotonin. The fact that it contains a prion-domain capable of propagating and stabilising a conformational change in the protein, and that this change equates to variant activities, has suggested an exciting answer to a hoary problem in neurobiology: the endurance of memories. Proteins and other cellular components are generally turned over in a matter of hours. How then can they be responsible for encoding memories that can last years? By CPEB undergoing a regulated prion-like polymerisation in response to synaptic transmission, a long-term memory of this stimulation can be stored. An equivalent CPEB in the fruit fly has recently been shown to be working in a similar way. It appears that this could be a more general mechanism for cellular memory storage in animal neurons.

A role for prions in memory is intriguing, as it hints at a reason why neurodegenerative diseases are so often associated with build ups of inactive mis-folded proteins. These ‘amyloid’ plaques are a feature of Alzheimer’s, Parkinson’s and Huntington’s diseases as well as the spongiform encephalopathies. Is this commonality a side effect of the brain normally permitting more regulated prion-like polymerisation events during memory formation?

The existence of self-replicating proteins, a new ‘epigenetic’ level of inheritance, has opened exciting new avenues of research. These new roles for prions in brain function and evolution could be just the tip of iceberg.

Shorter, J., & Lindquist, S. (2005). Prions as adaptive conduits of memory and inheritance Nature Reviews Genetics, 6 (6), 435-450 DOI: 10.1038/nrg1616

Halfmann, R., & Lindquist, S. (2010). Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits Science, 330 (6004), 629-632 DOI: 10.1126/science.1191081

See also,
Amyloid-like oligomers and long-term memory.

microDNAs: small mammalian extrachromosomal circular DNAs

A new paper in Science, reports the detection of a new species of DNA in mammalian cells: microDNA. microDNAs are extrachromosomal circular DNA molecules, generally 200-400bp long, derived from non-repetitive genomic sequence. microDNAs appear to arise from microdeletions occurring in the 5’ ends of genes. This data implies widespread genetic variation with respect to microdeletions between somatic cells in mammals. 

To identify sites of intramolecular homologous recombination that could lead to genetic mosaicism in mammalian brains, Shibata et al. searched for the extrachromosomal circular DNAs (eccDNA) that could be produced. DNA was purified from embryonic mouse brains and linear DNA was degraded with a specific exonuclease. The remaining fraction was then amplified with an unbiased non-PCR technique (multiple displacement amplification). The linear products were then sheared into 500bp fragments, cloned, and sequenced. The majority of the clones in the library included repeated sequences, consistent with the products of rolling circle amplification of small circular DNAs. When these repeated sequences were searched against the mouse genome, they were only found once, showing that they were not produced by repetitive sequence (eg. transposable elements). To prove that these sequences were indeed derived from circular DNA molecules, PCR using outward directed primers designed from the repeated sequences was performed on both extrachromosomal and chromosomal DNA. If the template DNA was circular, PCR amplification should occur, if linear, it shouldn’t. This was (generally) the case, proving the existence of a population of extrachromosomal circular DNAs, a few hundred base pairs long, derived from unique portions of the chromosomal genome.

To further explore the nature and extent of this population of DNA molecules, Shibata et al. went on to purify eccDNA from a range of embryonic and adult mouse tissues, and from mouse and human cancer cell lines. After amplification and the sequencing of the ends of the generated fragments, they found that tens of thousands of unique genomic sequences yield extrachromosomal circular DNAs. The eccDNA from mouse tissues ranged from 80-2000bp in length, but most were between 200-400bp. Lengths of ~200bp and ~400bp were enriched in the mouse brain and liver populations. A similar pattern was detected in human cancer cell lines, but in these eccDNA populations an additional pattern of length distribution peaks at a 150bp periodicity was detected. As in the earlier experiment, the circular DNAs mapped to unique positions in the genome. To differentiate this population of eccDNA from previously reported longer forms derived from repetitive sequence, the authors termed them microDNAs.

Electron micrograph showing double-stranded (left) and single-stranded (right) microDNAs.

The researchers went on to directly visualise microDNA molecules by electron microscopy. Using a technique that specifically labels single stranded DNA, they discovered both double-stranded and single-stranded microDNAs were present in approximately equal measure.

Bioinformatic analysis of the sources of microDNAs revealed high enrichment for 5’ UTRs, exons, and CpG islands (regions upstream of genes where cytosine residues in CG dinucleotides are not methylated), suggesting that microDNAs are commonly derived from the 5’ ends of genes. microDNAs also have a higher percentage GC content than the average for the genome (55% as opposed to 45%). In a relatively high proportion of microDNAs, the researchers detected short direct repeats of 2-15bp of microhomology at the starts and ends of the molecules.

microDNAs could potentially be created by excision from chromosomal DNA, by replication of short stretches of DNA, or by reverse transcription of RNA molecules. Shibata et al. selected two genomic loci that yielded microDNAs and found that microdeletions do occur in these regions in some cells. The lengths and GC content of the microdeletions that they identified were in line with those found in microDNAs. The majority of the microdeletions displayed short stretches of microhomology at their excised ends.

These short direct repeats at the start and ends of microDNAs, and at their presumptive source microdeletions, suggest two possibilities for microDNA generation. Regions of microhomology could cause the DNA replication process to stall and switch template. Incorrect repair processes would then lead to the release of a microDNA. Alternatively, microhomology mediated repair processes could lead to the excision of a microDNA by intramolecular homologous recombination. The 150bp length periodicity detected in the cancer cell line microDNAs is suggestive of a link to nucleosomes (in which ~150bp of chromosomal DNA are wrapped around the histone core). A link to the position of nucleosomes (either in tightly bound nucleosomes causing replication problems or in facilitating microDNA circularisation) may explain the enrichment of microDNAs from the 5’ends of genes. Another suggestion made to explain the origin of ss microDNA, is that they could be formed by displaced Okazaki fragments (the short sections of replicated DNA formed on the lagging strand). All of these ideas are ‘hand wavey stuff’ but exciting avenues for future experiments nonetheless. A couple of obvious counter-arguments to these suggestions would be that microhomologies were only detected in 37% of microDNAs, and that the 150bp periodicity was only found in the cancer cell line microDNAs. A combination of the above putative modes of microDNA generation could be taking place, and microDNAs may be a heterogeneous population of molecules (as the presence of ss and ds DNAs suggests).

Perhaps the most striking conclusion of this paper is that the widespread generation of microDNAs by microdeletions yields large amounts of genetic variation between somatic cells. This mosaicism may well lead to functional differences between cells. What are implications of this mosaicism? Do microDNAs have any specific functions? Or are they simply a product of defective replication/repair processes? Are microDNAs only found in mammalian cells? Or are they more widespread (the researchers didn’t observe any in yeast cells)? It will be exciting to see future research attempt to answer these questions.

Shibata, Y., Kumar, P., Layer, R., Willcox, S., Gagan, J., Griffith, J., & Dutta, A. (2012). Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues Science, 336 (6077), 82-86 DOI: 10.1126/science.1213307

On ICE: Integrative and Conjugative Elements.

Integrative and conjugative elements are bacterial mobile genetic elements that primarily reside in the host cell’s chromosome, yet have the ability to be transferred between cells by conjugation. ICEs can be considered as mosaic elements, combining features from other mobile elements: the integrative ability of bacteriophage or transposons, and the transfer mechanisms of conjugative plasmids. This mosaicism is reflected in modular structures: genes encoding the core functions of integration/excision, conjugation and regulation are generally found clustered together. As well as these core functions, ICEs often carry accessory genes that can bestow adaptive phenotypes on their hosts. Gene cassettes encoding antibiotic resistance, nitrogen fixation, virulence factors and various other functions have all been documented in ICEs. They are therefore important vectors for the horizontal dissemination of genetic information, facilitating rapid bacterial evolution.

Chromosomal integration and excision of ICEs is mediated by integrase (Int) enzymes. Most commonly integrases are tyrosine recombinases related to the well studied phage λ Int. They mediate site-specific recombination events between identical or near-identical sequences in the host and ICE genomes (termed attB and attP respectively). These integration events normally occur into tRNA genes. No definitive reason for this association of tyrosine recombinase mediated integration with tRNA genes is known, however tRNA genes evolve more slowly than protein coding genes, potentially broadening the possible host range. Other ICEs encode transposase family tyrosine recombinase Ints that have broader target sequence preferences. Members of the DDE transposase and serine recombinase families also serve as integrases in some ICEs. Before ICE transfer occurs, the element is excised and circularised. Excision of ICEs also requires Int activity, however the process is biased towards excision by ‘recombination directionality factors’ (RDFs). If chromosomal replication or cell devision occurs whilst the ICE is in the excised chromosomal state, the element could be lost from the cell. To prevent this ICEs (like plasmids) often encode addiction modules (toxin-antitoxin systems) that kill cells not inheriting the ICE.

Conjugative transfer occurs via the formation of a multiprotein apparatus that connects the donor and recipient cells: a type IV secretion system (T4SS). This consists of a membrane spanning secretion channel and often an extracellular pilus. The extrachromosomal ICE DNA is first nicked at the origin of transfer (oriT) by a relaxase (MOB) enzyme. Rolling circle replication is then initiated. MOB remains bound to the displaced single-stranded DNA and this nucleoprotein complex is targeted to the T4SS by a coupling protein (T4CP). Rather than using a T4SS, some ICEs are transferred between cells using FtsK-like DNA translocase pumps (in this case dsDNA is transferred). After transfer, the ssDNA ICE is replicated into dsDNA and integrated into the recipient cell’s chromosome. The ICE in the donor cell is also converted into dsDNA and re-integrated into the genome.

ICE transmission is under the control of networks responsive to environmental stimuli. For instance, transfer of the SXT-R391 family of ICEs is controlled by SelR, a homologue of the λ repressor CI. Regulation occurs by a similar mechanism as that controlling the λ switch from lysogeny to lysis. As with CI, SetR repression can be relieved by the action of RecA, the main effector of the ‘SOS’ response to DNA damage. Other ICEs transmission have been shown to be under the control of quorum sensing networks.

A recent bioinformatic study of ICE prevalence and diversity identified ICEs by finding clustered conjugative apparatus modules (Guglielmini et al). If these were found on chromosomal locations they were defined as belonging to ICEs, whilst those on extra-chromosomal elements were considered conjugative plasmids. No reference was made to the presence of integrases. Within this definition ICEs were more common than conjugative plasmids: 18% of sequenced prokaryotic genomes contained at least one ICE as opposed to 12% possessing conjugative plasmids. ICEs are generally defined as not being capable of autonomous extra-chromosomal replication and maintenance. This is opposed to conjugative plasmids that include replication origins and systems. However, this definition is not watertight, as there appear to be various exceptions. Likewise, conjugative plasmids can be integrated chromosomally, either by homologous recombination at repeat sequences, or by site-specific recombination events. These elements therefore exist on a spectrum. Phylogenetic analysis of VirB4 genes (an ATPase component of T4SS) shows that ICEs and conjugative plasmids do not segregate as monophyletic clades. Instead they are intermingled throughout the tree, suggesting that conjugative plasmids often become ICEs and vice versa. Guglielmini et al. therefore consider them as two sides of the same coin. If selection pressures are strong enough though, ICEs can be stabilised as chromosomal structures for long periods of time.

A striking example of the potency and evolutionary importance of ICEs is found in the genomes of the obligate intracellular bacterial family Rickettsiales. One third of the genome of Orientia tsutsugamushi (the mite borne causative agent of scrub typhus) is made up of degenerate copies of an ICE named RAGE (Rickettsiales amplified genetic element). Multiple invasions of RAGE have also configured the genome of a Rickettsial endosymbiont of a deer tick (REIS). In this case RAGEs have acted as hotspots for recombination and the insertion of other mobile elements, leading to the insertion of clusters of novel horizontally transferred genes (a process termed piggybacking). These two Rickettsiales species have especially large genomes for obligate intracellular bacteria, but it seems likely that RAGE has been important in the evolution of this entire clade.

See also: Expanding the Conjugative Realm

Wozniak, R., & Waldor, M. (2010). Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow Nature Reviews Microbiology, 8 (8), 552-563 DOI: 10.1038/nrmicro2382

Guglielmini, J., Quintais, L., Garcillán-Barcia, M., de la Cruz, F., & Rocha, E. (2011). The Repertoire of ICE in Prokaryotes Underscores the Unity, Diversity, and Ubiquity of Conjugation PLoS Genetics, 7 (8) DOI: 10.1371/journal.pgen.1002222

Nakayama, K., Yamashita, A., Kurokawa, K., Morimoto, T., Ogawa, M., Fukuhara, M., Urakami, H., Ohnishi, M., Uchiyama, I., Ogura, Y., Ooka, T., Oshima, K., Tamura, A., Hattori, M., & Hayashi, T. (2008). The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution DNA Research, 15 (4), 185-199 DOI: 10.1093/dnares/dsn011

Gillespie, J., Joardar, V., Williams, K., Driscoll, T., Hostetler, J., Nordberg, E., Shukla, M., Walenz, B., Hill, C., Nene, V., Azad, A., Sobral, B., & Caler, E. (2011). A Rickettsia Genome Overrun by Mobile Genetic Elements Provides Insight into the Acquisition of Genes Characteristic of an Obligate Intracellular Lifestyle Journal of Bacteriology, 194 (2), 376-394 DOI: 10.1128/JB.06244-11