This chapter reviews a selected number of representative topics relating to the genetics and molecular biology of halophilic archaebacteria and the author shall try to share his enthusiasm and excitement for some of the molecular curiosities that these organisms exhibit. The discussion focuses mostly on gene sequences, gene organization, gene regulation, and gene expression and less on protein structure and function. The methodologies employed are not mysterious or highly specialized; rather they are the standard methods of biochemistry and molecular biology what has become known collectively as recombinant DNA technology. The next phase in the development of the molecular biology of halophilic archaebacteria will hinge on progress in three areas: the total mapping, cloning, and ordering of genomic DNAs, definition of natural genetic exchange systems, and the development of cloning vector-transformation systems.

Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2'-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum, particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.

Archaea use C/D box sno-like RNAs (sRNAs) to guide precise 2u0027-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNAs are the most numerous archaeal small RNA class, most genomes have incomplete sRNA gene annotation because reliable, completely automated detection methods are unavailable. To study archaeal C/D box sRNA structure, function and evolution, we collected, predicted, and curated a comprehensive set of these sRNAs from six species within the crenarchaeal hyperthermophilic genus Pyrobaculum. We used high-throughput small RNA sequencing data, computational methods, and comparative genomics to curate 526 Pyrobaculum C/D box sRNAs and organized them into 110 families based on conservation of their guide sequences. Our comprehensive analysis provides a detailed narrative of C/D box sRNA evolutionary history, implications of target conservation on ribosome maturation, and sRNA impact on genome architecture. We find that in some cases the overlap of C/D box sRNAs with protein-encoding genes can abolish the ability of their guides to target rRNA and tRNAs. Numerous duplications and rearrangements of sRNA genes are illustrated, and some of these appear to employ a mechanism similar to the retrotransposition of C/D box sRNAs genes in eukaryotes. Finally, we used these annotations to train a new computational search model.

Background: In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no) RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.Results: We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.Conclusions: This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-Omethylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.

The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.

A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations.

In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kink-turn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 3'-terminal ACA sequence, and many contain only a single 3' guide region rather than the canonical 5' and 3' bipartite guides. The predicted sRNA structures contain guide sequences that exhibit strong complementarity to ribosomal RNA or transfer RNA. Most of the predicted targets of pseudouridine modification are structurally equivalent to those known in other species. One sRNA appears capable of guiding pseudouridine modification at positions U54 and U55 in most or all Pyrobaculum tRNAs. We experimentally tested seven predicted pseudouridine modifications in ribosomal RNA, and all but one was confirmed. The structural insights provided by this new set of Pyrobaculum sRNAs will augment existing models and may facilitate the identification and characterization of new guide sRNAs in other archaeal species.

Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus have uncovered a novel RNA-targeting variant of the CRISPR system. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genomic and transcriptomic view of the CRISPR arrays across six diverse species within the crenarchaeal genus Pyrobaculum. We present transcriptional data from each of four species in the genus (P. aerophilum, P. islandicum, P. calidifontis, P. arsenaticum), analyzing mature CRISPR-associated small RNA abundance from over 20 arrays. Within the genus, there is remarkable conservation of CRISPR array structure, as well as unique features that are have not been studied in other archaeal systems. These unique features include: a nearly invariant CRISPR promoter, conservation of direct repeat families, the 5' polarity of CRISPR-associated small RNA abundance, and a novel CRISPR-specific association with homologues of nurA and herA. These analyses provide a genus-level evolutionary perspective on archaeal CRISPR systems, broadening our understanding beyond existing non-comparative model systems.

During fast growth, the rrn P1 promoters of Escherichia coli operate at their maximum strength, but below their maximum activity (V(max)), since they are not saturated with RNA polymerase. Since higher concentrations of free RNA polymerase are expected to be found in strains carrying rrn deletions, we have analyzed reported electron micrographs of rrn operons from rrn deletion strains growing at maximal rates (at 37 degrees C) in LB medium [1]. We conclude that, in a strain with four of the seven rrn operons inactivated by partial deletions, transcripts are initiated at rrn P1 promoters 1.6-fold more rapidly than in the wild-type strain and the entirety of the rrn operon is transcribed at a 1.5-fold higher average elongation rate due to shortened pauses in the 16S and 23S regions. Under this condition, traffic congestion occurs in front of a pause site in the 5' leader region of the rrn operon near the beginning of the 16S gene; the congestion extends all the way back to the promoter, impedes promoter clearance and limits the promoter activity to one initiation per 0.56 s. This corresponds to a promoter activity of 107 transcripts/min and is assumed to be close to the V(max) of rrn P1 promoters.

The value of the rRNA chain elongation rate in bacteria is an important physiological parameter, as it affects not only the rRNA promoter activity but also the free-RNA polymerase concentration and thereby the transcription of all genes. On average, rRNA chains elongate at a rate of 80 to 90 nucleotides (nt) per s, and the transcription of an entire rrn operon takes about 60 s (at 37 degrees C). Here we have analyzed a reported distribution obtained from electron micrographs of RNA polymerase molecules along rrn operons in E. coli growing at 2.5 doublings per hour (S. Quan, N. Zhang, S. French, and C. L. Squires, J. Bacteriol. 187:1632-1638, 2005). The distribution exhibits two peaks of higher polymerase density centered within the 16S and 23S rRNA genes. An evaluation of this distribution indicates that RNA polymerase transcribes the 5' leader region at speeds up to or greater than 250 nt/s. Once past the leader, transcription slows down to about 65 nt/s within the 16S gene, speeds up in the spacer region between the 16S and 23S genes, slows again to about 65 nt/s in the 23S region, and finally speeds up to a rate greater than 400 nt/s near the end of the operon. We suggest that the slowing of transcript elongation in the 16S and 23S sections is the result of transcriptional pauses, possibly caused by temporary interactions of the RNA polymerase with secondary structures in the nascent rRNA.

This review begins by briefly presenting the history of research on the chemical composition and other parameters of cells of E. coli and S. enterica at different exponential growth rates. Studies have allowed us to determine the in vivo strength of promoters and have allowed us to distinguish between factor-dependent transcriptional control of the promoter and changes in promoter activity due to changes in the concentration of free functional RNA polymerase associated with different growth conditions. The total, or bulk, amounts of RNA and protein are linked to the growth rate, because most bacterial RNA is ribosomal RNA (rRNA). Since ribosomes are required for protein synthesis, their number and their rate of function determine the rate of protein synthesis and cytoplasmic mass accumulation. Many mRNAs made in the presence of amino acids have strong ribosome binding sites whose presence reduces the expression of all other active genes. This implies that there can be profound differences in the spectrum of gene activities in cultures grown in different media that produce the same growth rate. Five classes of growth-related parameters that are generally useful in describing or establishing the macromolecular composition of bacterial cultures are described in detail in this review. A number of equations have been reported that describe the macromolecular composition of an average cell in an exponential culture as a function of the culture doubling time and five additional parameters: the C- and D-periods, protein per origin (PO), ribosome activity, and peptide chain elongation rate.

Most organisms contain between 40 and 50 different transfer RNA (tRNA) molecules that read one or more of the 61 or 62 different sense codons through specific codon-anticodon interactions and position the appropriate amino acid for insertion into the growing polypeptide chain. Additional complexity in the tRNA processing and modification pathway occurs in cases where archaeal tRNA genes contain an intron. The chapter describes introns in archaeal transcripts. The genes for ribosomal RNA (rrn) are located in operons in the archaeal genome and are transcribed to produce multicistronic precursor RNAs. Two early studies used nuclease protection and primer extension assays to define the intermediates generated during processing of the primary rRNA transcript from two canonical rrn operons: the single-rrn operon in Halobacterium salinarum and the canonical rrnA operon in Haloarcula marismortui. The proportion of modified nucleotides in tRNA can approach 50% or more. The two most frequent modifications in RNA are ribose methylation and the pseudouridylation. In an attempt to distinguish between these two alternatives, actinomycin D was used to inhibit transcription and Northern hybridization was used to follow the decay of the various mRNA fragments. There was a strong correlation between fragment length and stability: the shorter the fragment, the longer the half-life. This correlation was used to argue that the transcript fragments are generated by endonucleotic cleavage rather than premature transcription termination, and that the distal sequences released following cleavage are selectively degraded.

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K-turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae-associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two-thirds of these sequences possessed K-turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5' or 3' ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.

Biochemical and informatics analyses conducted over the past few years have revealed the presence of a plethora of small non-coding RNAs in various species of Archaea. A large proportion of these RNAs contain a common structural motif called the RNA kink turn (K-turn). The best-characterized are the C/D box and the H/ACA box guide small (s)RNAs. Both contain the K-turn fold and require the binding of the L7Ae protein to stabilize the structure of this crucial motif. These sRNAs assemble with L7Ae and several other proteins into complex and dynamic ribonucleoprotein machines that mediate guide-directed ribose methylation or pseudouridylation to specific locations in ribosomal or transfer RNA. Analyses of new archaeal sRNA libraries have identified additional classes of novel sRNAs; many of these contain the RNA K-turn motif and suggest that the RNAs might function as ribonucleoprotein complexes. Some have characteristics of small interfering RNAs or of micro RNAs that have been implicated in the post-transcriptional control of gene expression, whereas others appear to be involved in protein translocation or in ribosomal RNA processing and ribosome assembly. A complete understanding of the structure of the K-turn motif and its contribution to various RNA-RNA and RNA-protein interactions will be absolutely essential to fully elucidate the biological organization, activity and function of these novel archaeal ribonucleoprotein machines.

The first pail of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (V-max/K-m) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation.

Archaea use ribonucleoprotein (RNP) machines similar to those found in the eukaryotic nucleolus to methylate ribose residues in nascent ribosomal RNA. The archaeal complex required for this 2'-O-ribose-methylation consists of the C/D box sRNA guide and three proteins, the core RNA-binding aL7a protein, the aNop56 protein and the methyltransferase aFib protein. These RNP machines were reconstituted in vitro from purified recombinant components, and shown to have methylation activity when provided with a simple target oligonucleotide, complementary to the sRNA guide sequence. To obtain a better understanding of the versatility and specificity of this reaction, the activity of reconstituted particles on more complex target substrates, including 5S RNA, tRNA(Gln) and 'double target' oligonucleotides that exhibit either direct or reverse complementarity to both the D' and D box guides, has been examined. The natural 5S and tRNA(Gln) substrates were efficiently methylated in vitro, as long as the complementarity between guide and target was about 10 base pairs in length, and lacked mismatches. Maximal activity of double guide sRNAs required that both methylation sites be present in cis on the target RNA.