Patrick P. Dennis

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C/D box small RNAs that guide 2′-O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2′-O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2′-O-methylation of ribose in a target RNA.

A guided tour: small RNA function in Archaea.

In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2′‐O‐ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis. Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature. The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs. The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature. We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2′‐O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.

Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates

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.

RNA‐modifying machines in archaea

It has been known for nearly half a century that coding and non‐coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non‐coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA‐containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small‐non‐coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

mRNA Composition and Control of Bacterial Gene Expression

The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of beta-galactosidase expressed from three different promoter-lacZ fusions (P(spc)-lacZ, P(RNAI)-lacZ, and P(RNAII)-lacZ) was measured (i) in a relA(+) background during exponential growth at different rates and (ii) in relA(+) and DeltarelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp.

Sequence Alignment and Evolutionary Comparison of the L10 Equivalent and L12 Equivalent Ribosomal Proteins from Archaebacteria, Eubacteria, and Eucaryotes

SummaryThe genes corresponding to the L10 and L12 equivalent ribosomal proteins (L10e and L12e) ofEscherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria,Halobacterium cutirubrum andSulfolobus solfataricus. The deduced amino acid sequences of the L10e and L12e proteins have been compared to each other and to available eubacterial and eucaryotic sequences. We have identified the hyman P0 protein as the eucaryotic L10e. The L10e proteins from the three kingdoms were found to be colinear. The eubacterial L10e protein is much shorter than the archaebacterial-eucaryotic proteins because of two large deletions, one internal and one at the carboxy terminus. The archaebacterial and eucaryotic L12e proteins were also colinear; the eubacterial protein is homologous to the archaebacterial and eucaryotic L12e proteins, but has suffered rearrangement through what appear to be gene fusion events. Intraspecies comparisons between L10e and L12e sequences indicate the archaebacterial and eucaryotic L10e proteins contain a partial copy of the L12e protein fused to their carboxy terminus. In the eubacteria most of this fusion has been removed by the carboxy terminal deletion. Within the L12e-derived region, a 26-amino acid-long internal modular sequence reiterated thrice in the archaebacterial L10e, twice in the eucaryotic L10e, and once in the eubacterial L10e was discovered. This modular sequence also appears to be present as a single copy in all L12e proteins and may play a role in L12e dimerization, L10e–L12e complex formation, and the function of L10e–L12e complex in translation. From these sequence comparisons a model depicting the evolutionary progression of the L10e and L12e genes and proteins from the primordial state to the contemporary archaebacterial, eucaryotic, and eubacterial states is presented.

RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria.

Abstract. A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits β and β′(rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (β and β′) showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits β and β′, A. pyrophilus was placed within the Gram-negative bacteria below the ε subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall–less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.

Characterization of the L11, L1, L10 and L12 equivalent ribosomal protein gene cluster of the halophilic archaebacterium Halobacterium cutirubrum.

We have cloned and characterized a 5.2 kb fragment of genomic Halobacterium cutirubrum DNA encoding two potential proteins of unknown function (ORF and NAB) and four proteins which are equivalent to the L11, L1, L10 and L12 ribosomal proteins of Escherichia coli (L11e, L1e, L10e and L12e). The ribosomal protein genes are clustered in the same order as that in E. coli although the transcription pattern differs. Transcripts characterized include (i) abundant monocistronic L11e and tricistronic L1e‐L10e‐L12e transcripts; (ii) less abundant bicistronic NAB‐L11e and monocistronic NAB transcripts and (iii) a very rare ORF monocistronic transcript. The consensus sequence in the promoter region is TTCGA … 4‐10 nucleotides … TTAA … 25‐26 nucleotides … initiation site; termination generally occurs on poly(T) tracts following GC‐rich regions. Poly(T) tracts in the sense strands within coding regions are notably absent; this is probably related to their participation in transcription termination and to the fact that these ribosomal protein genes are highly expressed and stoichiometrically balanced. In the third position of the codons G or C is utilized 87% of the time. The 74 nt long untranslated leader of the L1e‐L10e‐L12e transcript contains a region that has a sequence and structure almost identical to a region within the binding domain for the L1e protein in 23S rRNA and highly similar to the E. coli L11‐L1 mRNA leader sequence that has been implicated in autogenous translational regulation. Other transcripts are initiated at or adjacent to the ATG translation initiation codon.

The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli B

SummaryThe effects of a partial restriction of valyl-tRNA aminoacylation on the synthesis of aminoacyl-tRNA synthetases, ribosomal proteins, and other translation and transcription proteins were examined in otherwise isogenic stringent (relA+) and relaxed (relA1) derivatives of E. coli B. The synthesis of individual ribosomal proteins, elongation factor G, and to a lesser extent elongation factors Tu and Ts, and the valyl- and arginyl-tRNA synthetases was found to be subject to the influence of the stringent control system. The synthesis of the α and β subunits of RNA polymerase and several of the aminoacyl-tRNA synthetases, in contrast, is either not subject to the influence of the stringent control system, or is subject to additional regulatory constraints.

Transcription patterns of adjacent segments on the chromosome of Escherichia coli containing genes coding for four 50 S ribosomal proteins and the β and β′ subunits of RNA polymerase

Abstract The transcription of four distinct segments of the bacterial chromosome has been examined under different conditions of steady-state growth using specific RNA-DNA hybridization assays. The genome segments contain, respectively: (1) a cluster of genes coding for four ribosomal proteins, (2) the gene coding for the β subunit of RNA polymerase, (3) the gene coding for the β′ subunit of RNA polymerase and (4) a cluster of genes coding for 15 ribosomal proteins and the α subunit of RNA polymerase. The first three segments are contiguous and located near 88 minutes, whereas the fourth segment is distinctly separate and located near 72 minutes on the Escherichia coli linkage map. The amount of RNA (measured as a fraction of the total cellular RNA) which hybridized to the DNA segments coding for ribosomal proteins was found to increase with the steady-state growth rate of the bacterium. In contrast, the amount of RNA that was homologous to the genes coding for the β and β′ subunits of RNA polymerase was invariant with the growth rate. The amount of β′ messenger RNA was apparently greater than the amount of β mRNA, suggesting that β′ mRNA is more stable than β mRNA during the exponential phase growth. The amount and frequency of transcription of these DNA segments was determined by hybridization with pulse-labeled RNA. The results of these experiments surprisingly indicated that the four segments were transcribed proportionately and independent of the bacterial growth rate. The ribosomal protein gene clusters at 88 minutes and 72 minutes were transcribed with about equal frequency; this indicates that the transcription of distinctly separate gene clusters can be regulated co-ordinately. The DNA segments coding for the β and β′ subunits of RNA polymerase were transcribed with equal frequency, and their transcription was co-ordinate with, but only one-fourth or one-fifth as frequent as transcription of the adjacent ribosomal protein genes. This observation is consistent with the idea that the genes coding for the β and β′ subunits are cotranscribed, and further suggests that regulation of the β and β′ subunit genes is intimately related to the regulation of the adjacent ribosomal protein genes.