RESUMO
In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved ß-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.
Assuntos
Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Proteínas de Bactérias/biossíntese , Flavobacterium/metabolismo , Motivos de Nucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MOTIVATION: Ribosome profiling has been widely used to study translation in a genome-wide fashion. It requires deep sequencing of ribosome protected mRNA fragments followed by mapping of fragments to the reference genome. For applications such as identification of ribosome pausing sites, it is not enough to map a fragment to a given gene, but the exact position of the ribosome represented by the fragment must be identified for each mRNA fragment. The assignment of the correct ribosome position is complicated by the broad length distribution of the ribosome protected fragments caused by the known sequence bias of micrococcal nuclease (MNase), the most widely used nuclease for digesting mRNAs in bacteria. Available mapping algorithms suffer from either MNase bias or low accuracy in characterizing the ribosome pausing kinetics. RESULTS: In this paper, we introduce a new computational method for mapping the ribosome protected fragments to ribosome locations. We first develop a mathematical model of the interplay between MNase digestion and ribosome protection of the mRNAs. We then use the model to reconstruct the ribosome occupancy profile on a per gene level. We demonstrate that our method has the capability of mitigating the sequence bias introduced by MNase and accurately locating ribosome pausing sites at codon resolution. We believe that our method can be broadly applied to ribosome profiling studies on bacteria where codon resolution is necessary. AVAILABILITY AND IMPLEMENTATION: Source code implementing our approach can be downloaded under GPL3 license at http://bioserv.mps.ohio-state.edu/RiboProP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Ribossomos , Algoritmos , Códon , Biossíntese de Proteínas , RNA MensageiroRESUMO
RNA forms elaborate secondary structures through intramolecular base pairing. These structures perform critical biological functions within each cell. Due to the availability of a polynomial algorithm to calculate the partition function over these structures, they are also a suitable system for the statistical physics of disordered systems. In this model, below the denaturation temperature, random RNA secondary structures exist in one of two phases: a strongly disordered, low-temperature glass phase and a weakly disordered, high-temperature molten phase. The probability of two bases to pair decays with their distance with an exponent 3/2 in the molten phase and about 4/3 in the glass phase. Inspired by previous results from a renormalized field theory of the glass transition separating the two phases, we numerically study this transition. We introduce distinct order parameters for each phase that both vanish at the critical point. We finally explore the driving mechanism behind this transition.
Assuntos
Vidro/química , Conformação de Ácido Nucleico , Transição de Fase , RNA/química , Sequência de Bases , Modelos Moleculares , RNA/genética , Temperatura de TransiçãoRESUMO
OBJECTIVES: In > 50% of cancers tumor development involves the early loss of Fhit (fragile histidine triad) protein expression, yet the mechanistic pathway(s) by which Fhit mediates its tumor suppressor functions are not fully understood. Earlier attempts to identify a Fhit-deficient gene expression profile relied on total cellular RNA and microarray analysis. The data here used RNA sequencing (RNA-Seq) of Fhit-negative and Fhit-positive cells as proof of principle for the impact of Fhit on specific mRNAs, and to lay the foundation for a study using ribosome profiling to identify mRNAs whose translation is affected by FHIT loss. DATA DESCRIPTION: RNA-Seq was performed on RNA from lines of Fhit-expressing and Fhit-deficient lung cancer cells. This identified changes in the levels of mRNAs for a number of cell survival and cell cycle progression genes. Polysome profile analysis performed on cytoplasmic extracts from Fhit-negative and Fhit-positive cells showed changes in the sedimentation of select mRNAs consistent with changes in translation efficiency. The impact of differential Fhit expression on the turnover of selected cancer-linked mRNAs was determined by RT-qPCR of cytoplasmic RNA isolated at intervals after treating cells with a transcription inhibitor.