Ensemble simulations: folding, unfolding and misfolding of high‐efficiency transfer‐messenger RNA pseudoknot

ABSTRACT The effect of deletion of the operator-distal genes of the trp operon, including the trpE-trpD intercistronic punctuation point, on the degree of transcriptional polarity (in this case the effect of a nonsense mutation on the level of mRNA from the distal part of the very gene where the mutation is located) was investigated. Double mutants which contain a nonsense mutation and a deletion in trpE were constructed, and the degree of transcriptional polarity was estimated by the decrease in messenger RNA for the operator-distal trpE beyond the nonsense mutation, as well as by the production of truncated messenger RNA for the region of trpE proximal to the nonsense mutation. The content of mRNA of operator-distal trpE and the size of the mRNA of operator-proximal trpE of the double mutants show that transcriptional polarity is not relaxed as a function of distance of the nonsense mutation from the operator-distal end of the trpE segment (at which the subsequent high efficiency translational initiation signal has been deleted). These findings are consistent with the conclusion that the degree of polarity depends on the distance of the nonsense mutation fro mthe subsequent translation initiation signal, but not on its distance from the operator-distal end, including possible translational or transcriptional termination signals

Download Full-text

An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.2.713 ◽

1992 ◽

Vol 89 (2) ◽

pp. 713-717 ◽

Cited By ~ 89

Author(s):

M. Chamorro ◽

N. Parkin ◽

H. E. Varmus

Keyword(s):

Messenger Rna ◽

Ribosomal Frameshifting ◽

Highly Efficient ◽

Rna Pseudoknot

Download Full-text

Ribosomal pausing during translation of an RNA pseudoknot.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6931 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6931-6940 ◽

Cited By ~ 149

Author(s):

P Somogyi ◽

A J Jenner ◽

I Brierley ◽

S C Inglis

Keyword(s):

High Efficiency ◽

Mutational Analysis ◽

Base Pairs ◽

Stem Loop ◽

Pseudoknot Structure ◽

Stem Loop Structure ◽

Dead End ◽

Rna Pseudoknot ◽

Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

Download Full-text

Development of PAR-CLIP to analyze RNA-protein interactions in prokaryotes

10.1101/855189 ◽

2019 ◽

Author(s):

Sandeep Ojha ◽

Chaitanya Jain

Keyword(s):

Protein Interactions ◽

Messenger Rna ◽

High Efficiency ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nucleoside Analogs ◽

E Coli ◽

Rna Targets ◽

Genome Wide ◽

Established Technique

AbstractThe ability to identify RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as “Crosslinking and Immunoprecipitation” (CLIP) has revolutionized the genome-wide discovery of RNA targets. Among the different versions of CLIP developed, the incorporation of photoactivable nucleoside analogs into cellular RNA has proven to be especially valuable, allowing for high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Although PAR-CLIP has become an established technique for use in eukaryotes, it has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we first investigated whether 4-thiouridine (4SU), a photoactivable nucleoside, can be incorporated into E. coli RNA. After determining 4SU incorporation into RNA, we developed suitable conditions for crosslinking of proteins in E. coli cells and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA) - messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq. Based on our results, PAR-CLIP represents an improved method to identify the RNAs recognized by RNA-BPs in prokaryotes.

Download Full-text

Pseudoknot Structures in Retroviral and Bacteriophage Messenger RNA: a Family of Structurally Related RNA Pseudoknots

Microscopy and Microanalysis ◽

10.1017/s1431927600007364 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 95-96

Author(s):

D.W. Hoffman ◽

Z. Du ◽

J.A. Holland ◽

M.R. Hansen ◽

Y. Wang ◽

...

Keyword(s):

Messenger Rna ◽

Three Dimensional ◽

Structural Features ◽

Dimensional Structure ◽

Three Dimensional Model ◽

Single Nucleotide ◽

Rna Pseudoknots ◽

Gene 32 ◽

Rna Pseudoknot ◽

Bacteriophage T2

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three-dimensional structure of an RNA pseudoknot with a sequence corresponding to the 5' end region of the gene 32 messenger RNA of bacteriophage T2. NMR results show that the pseudoknot contains two coaxial A-form helical stems connected by two loops. One of the loops consists of a single nucleotide, which spans the major groove of the seven base pair helical stem 2. The second loop consists of 7 nucleotides, and spans the minor groove of stem 1. A three-dimensional model of the pseudoknot that is consistent with the NMR data will be presented, and features that are likely to be important for stabilizing the pseudoknot structure will be described.A combination of NMR and phylogenetic methods were used to characterize the structural features of RNA pseudoknots that are associated with frameshift and readthrough sites within the retroviral gag-pro messenger RNA. The majority of the retroviral frameshift and readthrough sites were found to be followed by nucleotide sequences that have the potential to form pseudoknots with structures that are remarkably similar to that of the bacteriophage T2 gene 32 mRNA.

Download Full-text

Thermodynamics of Folding of the RNA Pseudoknot of the T4 Gene32Autoregulatory Messenger RNA†

Biochemistry ◽

10.1021/bi9527348 ◽

1996 ◽

Vol 35 (13) ◽

pp. 4176-4186 ◽

Cited By ~ 22

Author(s):

Huawei Qiu ◽

Kumar Kaluarachchi ◽

Zhihua Du ◽

David W. Hoffman ◽

David P. Giedroc

Keyword(s):

Messenger Rna ◽

Rna Pseudoknot

Download Full-text

Regulators of Viral Frameshifting: More Than RNA Influences Translation Events

Annual Review of Virology ◽

10.1146/annurev-virology-012120-101548 ◽

2020 ◽

Vol 7 (1) ◽

pp. 219-238

Author(s):

Wesley D. Penn ◽

Haley R. Harrington ◽

Jonathan P. Schlebach ◽

Suchetana Mukhopadhyay

Keyword(s):

Messenger Rna ◽

Protein Production ◽

Productive Infection ◽

Cis Elements ◽

Stem Loop ◽

Viral Translation ◽

Ribosomal Frameshifting ◽

Evolutionarily Conserved ◽

Rna Pseudoknot ◽

New Protein

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand.

Download Full-text