Transcriptional accuracy modeling suggests two-step proofreading by RNA polymerase.

We suggest a novel two-step proofreading mechanism with two sequential rounds of proofreading selection in mRNA transcription. It is based on the previous experimental observations that the proofreading RNA polymerase cleaves off transcript fragments of at least 2 nt and that transcript elongation after a nucleotide misincorporation is anomalously slow. Taking these results into account, we extend the description of the accuracy of template guided nucleotide selection beyond previous models of RNA polymerase-dependent DNA transcription. The model derives the accuracy of initial and proofreading base selection from experimentally estimated nearest-neighbor parameters. It is also used to estimate the small accuracy enhancement of polymerase revisiting of previous positions following transcript cleavage.

Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs.

The ribosome uses initial and proofreading selection of aminoacyl-tRNAs for accurate protein synthesis. Anomalously high initial misreading in vitro of near-cognate codons by tRNA(His) and tRNA(Glu) suggested potential error hotspots in protein synthesis, but in vivo data suggested their partial neutralization. To clarify the role of proofreading in this error reduction, we varied the Mg(2+) ion concentration to calibrate the total accuracy of our cell-free system to that in the living Escherichia coli cell. We found the total accuracy of tRNA selection in our system to vary by five orders of magnitude depending on tRNA identity, type of mismatch, and mismatched codon position. Proofreading and initial selection were positively correlated at high, but uncorrelated at low initial selection, suggesting hyperactivated proofreading as a means to neutralize potentially disastrous initial selection errors.

Thermodynamic modeling of variations in the rate of RNA chain elongation of E. coli rrn operons.

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.

The recombination landscape of the zebra finch Taeniopygia guttata genome.

Understanding the causes and consequences of variation in the rate of recombination is essential since this parameter is considered to affect levels of genetic variability, the efficacy of selection, and the design of association and linkage mapping studies. However, there is limited knowledge about the factors governing recombination rate variation. We genotyped 1920 single nucleotide polymorphisms in a multigeneration pedigree of more than 1000 zebra finches (Taeniopygia guttata) to develop a genetic linkage map, and then we used these map data together with the recently available draft genome sequence of the zebra finch to estimate recombination rates in 1 Mb intervals across the genome. The average zebra finch recombination rate (1.5 cM/Mb) is higher than in humans, but significantly lower than in chicken. The local rates of recombination in chicken and zebra finch were only weakly correlated, demonstrating evolutionary turnover of the recombination landscape in birds. The distribution of recombination events was heavily biased toward ends of chromosomes, with a stronger telomere effect than so far seen in any organism. In fact, the recombination rate was as low as 0.1 cM/Mb in intervals up to 100 Mb long in the middle of the larger chromosomes. We found a positive correlation between recombination rate and GC content, as well as GC-rich sequence motifs. Levels of linkage disequilibrium (LD) were significantly higher in regions of low recombination, showing that heterogeneity in recombination rates have left a footprint on the genomic landscape of LD in zebra finch populations.

[Mycoplasma genitalium should be suspected in unspecific urethritis and cervicitis. A study from Vasterbotten confirms the high prevalence of the bacteria]. / Mycoplasma genitalium bör misstänkas vid ospecifik uretrit och cervicit. Studie från Västerbotten bekräftar den höga prevalensen av bakterien.

The microbe Mycoplasma genitalium has in several studies been proposed as an individual cause of non-gonococcal urethritis (NGU) in men, and has been associated with pelvic inflammatory disease (PID) and salpingitis. The prevalence of M genitalium has generally been 50-90% of the prevalence of C trachomatis, and this seems to be the case in Sweden as well. This is the first study of the pathogenesis and prevalence of M genitalium in northern Sweden. In total 823 samples, 340 from women and 483 from men, were screened for M genitalium by using a PCR method. Thirtythree (4.0%) patients, 13 (3.8%) women and 20 (4.1%) men, were infected by M genitalium. In the same group 60 (7.3%) patients, 16 (4.7%) women and 44 (9.1%) men, were infected by Chlamydia trachomatis. None of the 22 patients that were tested after treatment with azitromycin was still infected.

DNA template dependent accuracy variation of nucleotide selection in transcription.

It has been commonly assumed that the effect of erroneous transcription of DNA genes into messenger RNAs on peptide sequence errors are masked by much more frequent errors of mRNA translation to protein. We present a theoretical model of transcriptional accuracy. It uses experimentally estimated standard free energies of double-stranded DNA and RNA/DNA hybrids and predicts a DNA template dependent transcriptional accuracy variation spanning several orders of magnitude. The model also identifies high-error as well a high-accuracy transcription motifs. The source of the large accuracy span is the context dependent variation of the stacking free energy of pairs of correct and incorrect base pairs in the ever moving transcription bubble. Our model predictions have direct experimental support from recent single molecule based identifications of transcriptional errors in the C. elegans transcriptome. Our conclusions challenge the general view that amino acid substitution errors in proteins are mainly caused by translational errors. It suggests instead that transcriptional error hotspots are the dominating source of peptide sequence errors in some DNA template contexts, while mRNA translation is the major cause of protein errors in other contexts.