Riboregulator

Last updated October 21, 2023

In molecular biology, a riboregulator is a ribonucleic acid (RNA) that responds to a signal nucleic acid molecule by Watson-Crick base pairing. A riboregulator may respond to a signal molecule in any number of manners including, translation (or repression of translation) of the RNA into a protein, activation of a ribozyme, release of silencing RNA (siRNA), conformational change, and/or binding other nucleic acids. Riboregulators contain two canonical domains, a sensor domain and an effector domain. These domains are also found on riboswitches, but unlike riboswitches, the sensor domain only binds complementary RNA or DNA strands as opposed to small molecules. Because binding is based on base-pairing, a riboregulator can be tailored to differentiate and respond to individual genetic sequences and combinations thereof.

Types of riboregulators

Translational riboregulator

Translational riboregulators regulate the ability of a ribosome complex to scan, assemble, and/or translate an RNA molecule into a protein. In translational riboregulators, the RNA molecule is repressed or de-repressed depending on the secondary structure of the RNA molecule. Signal-responsive structures are usually introduced into the 5′ untranslated region (5′ UTR) of the RNA molecules using standard molecular biological techniques.

As discovered by Marilyn Kozak, the small (40S) ribosome complex scans an RNA molecule from 5′ untranslated region to the start codon. When the complex encounters secondary structure, it must melt the structure to reach the start codon or it will fall off the molecule.^[3] The complex moves along through the untranslated region until it stalls just prior to reaching the start codon because it encounters a highly conserved sequence (a Kozak consensus sequence in eukaryotes, or Shine-Dalgarno sequence in prokaryotes). The stalled complex then combines with the large ribosome (60S) to begin translating the RNA into protein.

Lechner invented the first riboregulator in 1991.^[2] Lechner used a self pairing stem-loop which inhibited translation of a prokaryotic RNA, unless a complementary RNA sequence (anti-inhibitor) was present. In 1997, Black engineered the first eukaryotic riboregulator using antisense molecules to prevent translation (Nucline RNA).^[1] In the Nucline system, antisense molecules block translation unless removed via competitive hybridization and strand-displacement by specific signal RNA sequences such as HIV RNA and oncogene RNA.^[4] In 2003, Black demonstrated that Nucline RNA could perform Boolean and arithmetic operations (If-then-else, AND gate, OR gate and "Molar concentration of x > molar concentration of y" calculations ) in HIV infected monocytes and breast cancer cells.^[5]^[6]

In 2004, Isaacs et al. modified Lechner's original system to demonstrate both trans and cis repression in a prokaryotic cells.^[7] Bayer & Smolke further developed a translational riboregulator in 2005 that could respond to small molecules creating a hybrid riboswitch/riboregulator molecule, termed an anti-switch.^[8] In an anti-switch, the presence of a small organic molecule binds an aptamer sequence in the RNA molecule which unmasks an otherwise sequestered antisense sequence, which can bind and block target RNA translation.

Ribozyme riboregulator

Ribozyme riboregulators regulate the ability of a catalytic RNA molecule to cleave a target nucleic acid sequence. In ribozyme riboregulators, a hammerhead ribozyme RNA molecule is activated or inactivated depending on the change of the secondary structure induced by hybridizing a signal molecule such as a cognate DNA or RNA sequence. In 2008, Win & Smolke designed a ribozyme regulator that could function in yeast cells that carried out Boolean operations similar to the earlier translational riboregulators, including AND, NAND, NOR, and OR gates.^[9]

RNAi-based riboregulator

RNAi riboregulators are small interfering RNAs which respond to a signal input such as complementary hybridization with a DNA or RNA molecule. The presence or absence of a target molecule determines whether the siRNA downregulates gene expression. In 2007, Rinaudo et al. demonstrated that RNAi based riboregulators can also perform Boolean operations in cells.^[10]

pH riboregulator

pH riboregulators regulate gene expression in response to pH changes. The only known pH riboregulator upregulates the alx gene in E. coli when in alkaline conditions, particularly above pH 8.^[11] The pH riboregulator was found, through experimentation, to reside in the 5′ UTR of the alx gene.^[12] When in pH 7 or below, the pH riboregulator element (PRE) exists in an inactive 'N' state; in alkali conditions, RNA polymerase controls the folding of this ncRNA to its 'H' active form. The ribosome binding site of the alx gene is now exposed and the 30s subunit is allowed to bind.^[12]

Uses of riboregulators

Currently, riboregulators have found utility in the area of synthetic biology and personalized medicine. In synthetic biology, riboregulators can be used to regulate bacterial responses and probe gene regulatory networks. Undergraduate systems biology students use standardized riboregulators to compete in an annual bio-engineering competition (The international Genetically Engineered Machine competition, iGEM).^[13] In addition, because the various riboregulators can be tailored to respond to complex biological signals, riboregulators offer the promise of individually tailored RNA based drugs which respond to genes, small molecules and proteins in individuals cells.

Related Research Articles

<span class="mw-page-title-main">Messenger RNA</span> RNA that is read by the ribosome to produce a protein

In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.

Protein biosynthesis is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

<span class="mw-page-title-main">Three prime untranslated region</span> Sequence at the 3 end of messenger RNA that does not code for product

In molecular genetics, the three prime untranslated region (3′-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3′-UTR often contains regulatory regions that post-transcriptionally influence gene expression.

In molecular biology, a reading frame is a way of dividing the sequence of nucleotides in a nucleic acid molecule into a set of consecutive, non-overlapping triplets. Where these triplets equate to amino acids or stop signals during translation, they are called codons.

<span class="mw-page-title-main">Morpholino</span> Chemical compound

A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule used in molecular biology to modify gene expression. Its molecular structure contains DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function.

The 5′ untranslated region is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. While called untranslated, the 5′ UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA. In many organisms, however, the 5′ UTR is completely untranslated, instead forming a complex secondary structure to regulate translation.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.

The Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. Regarded as the optimum sequence for initiating translation in eukaryotes, the sequence is an integral aspect of protein regulation and overall cellular health as well as having implications in human disease. It ensures that a protein is correctly translated from the genetic message, mediating ribosome assembly and translation initiation. A wrong start site can result in non-functional proteins. As it has become more studied, expansions of the nucleotide sequence, bases of importance, and notable exceptions have arisen. The sequence was named after the scientist who discovered it, Marilyn Kozak. Kozak discovered the sequence through a detailed analysis of DNA genomic sequences.

In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context, sense may have slightly different meanings. For example, negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.

Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-ring means that there will be a 5′ end, which frequently contains a phosphate group attached to the 5′ carbon of the ribose ring, and a 3′ end, which typically is unmodified from the ribose -OH substituent. In a DNA double helix, the strands run in opposite directions to permit base pairing between them, which is essential for replication or transcription of the encoded information.

<span class="mw-page-title-main">Untranslated region</span> Non-coding regions on either end of mRNA

In molecular genetics, an untranslated region refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 5' side, it is called the 5' UTR, or if it is found on the 3' side, it is called the 3' UTR. mRNA is RNA that carries information from DNA to the ribosome, the site of protein synthesis (translation) within a cell. The mRNA is initially transcribed from the corresponding DNA sequence and then translated into protein. However, several regions of the mRNA are usually not translated into protein, including the 5' and 3' UTRs.

Ribosomal frameshifting, also known as translational frameshifting or translational recoding, is a biological phenomenon that occurs during translation that results in the production of multiple, unique proteins from a single mRNA. The process can be programmed by the nucleotide sequence of the mRNA and is sometimes affected by the secondary, 3-dimensional mRNA structure. It has been described mainly in viruses, retrotransposons and bacterial insertion elements, and also in some cellular genes.

Ribosome profiling, or Ribo-Seq, is an adaptation of a technique developed by Joan Steitz and Marilyn Kozak almost 50 years ago that Nicholas Ingolia and Jonathan Weissman adapted to work with next generation sequencing that uses specialized messenger RNA (mRNA) sequencing to determine which mRNAs are being actively translated. A related technique that can also be used to determine which mRNAs are being actively translated is the Translating Ribosome Affinity Purification (TRAP) methodology, which was developed by Nathaniel Heintz at Rockefeller University. TRAP does not involve ribosome footprinting but provides cell type-specific information.

Marilyn S. Kozak is an American professor of biochemistry at the Robert Wood Johnson Medical School. She was previously at the University of Medicine and Dentistry of New Jersey before the school was merged. She was awarded a PhD in microbiology by Johns Hopkins University studying the synthesis of the Bacteriophage MS2, advised by Daniel Nathans. In her original faculty job proposal, she sought to study the mechanism of eukaryotic translation initiation, a problem long thought to have already been solved by Joan Steitz. While in the Department of Biological Sciences at University of Pittsburgh, she published a series of studies that established the scanning model of translation initiation and the Kozak consensus sequence. Her current research interests are unknown as her last publication was in 2008.

Translation regulation by 5′ transcript leader cis-elements is a process in cellular translation.

This glossary of genetics is a list of definitions of terms and concepts commonly used in the study of genetics and related disciplines in biology, including molecular biology, cell biology, and evolutionary biology. It is intended as introductory material for novices; for more specific and technical detail, see the article corresponding to each term. For related terms, see Glossary of evolutionary biology.

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