Attenuator (genetics)

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In genetics, attenuation is a regulatory mechanism for some bacterial operons that results in premature termination of transcription. The canonical example of attenuation used in many introductory genetics textbooks, [1] is ribosome-mediated attenuation of the trp operon. Ribosome-mediated attenuation of the trp operon relies on the fact that, in bacteria, transcription and translation proceed simultaneously. Attenuation involves a provisional stop signal (attenuator), located in the DNA segment that corresponds to the leader sequence of mRNA. During attenuation, the ribosome becomes stalled (delayed) in the attenuator region in the mRNA leader. Depending on the metabolic conditions, the attenuator either stops transcription at that point or allows read-through to the structural gene part of the mRNA and synthesis of the appropriate protein.

Contents

Attenuation is a regulatory feature found throughout Archaea and Bacteria causing premature termination of transcription. [2] Attenuators are 5'-cis acting regulatory regions which fold into one of two alternative RNA structures which determine the success of transcription. [3] The folding is modulated by a sensing mechanism producing either a Rho-independent terminator, resulting in interrupted transcription and a non-functional RNA product; or an anti-terminator structure, resulting in a functional RNA transcript. There are now many equivalent examples where the translation, not transcription, is terminated by sequestering the Shine-Dalgarno sequence (ribosomal binding site) in a hairpin-loop structure. While not meeting the previous definition of (transcriptional) attenuation, these are now considered to be variants of the same phenomena [3] and are included in this article. Attenuation is an ancient regulatory system, prevalent in many bacterial species providing fast and sensitive regulation of gene operons and is commonly used to repress genes in the presence of their own product (or a downstream metabolite). [3]

Classes of attenuators

Attenuators may be classified according to the type of molecule which induces the change in RNA structure. It is likely that transcription-attenuation mechanisms developed early, perhaps prior to the archaea/bacteria separation and have since evolved to use a number of different sensing molecules (the tryptophan biosynthetic operon has been found to use three different mechanisms in different organisms.) [2]

Ribosome-mediated attenuation

In this situation RNA polymerase is dependent on (lagging) ribosome activity; if the ribosome pauses due to insufficient charged tRNA then the anti-terminator structure is favoured. The canonical attenuator example of the trp operon uses this mechanism in E. coli. Similar regulatory mechanisms have been found in many amino acid biosynthetic operons. [4] [5]

Small-molecule-mediated attenuation (riboswitches)

Riboswitch sequences (in the mRNA leader transcript) bind molecules such as amino acids, nucleotides, sugars, vitamins, metal ions and other small ligands [3] which cause a conformational change in the mRNA. Most of these attenuators are inhibitory and are employed by genes for biosynthetic enzymes or transporters [3] whose expression is inversely related to the concentration of their corresponding metabolites. Example- Cobalamine biosynthesis, Cyclic AMP-GMP switch, lysin biosynthesis, glycine biosynthesis, fluroide switch etc.

T-boxes

These elements are bound by specific uncharged tRNAs and modulate the expression of corresponding aminoacyl-tRNA synthetase operons. [2] High levels of uncharged tRNA promote the anti-terminator sequence leading to increased concentrations of charged tRNA. These are considered by some to be a separate family of riboswitches [6] but are significantly more complex than the previous class of attenuators.

Protein-mediated attenuation

Protein-RNA interactions may prevent or stabilize the formation of an anti-terminator structure. [2] .. karima eric discovery

RNA thermometers

Temperature dependent loop formations introduce temperature-dependence in the expression of downstream operons. All such elements act in a translation-dependent manner by controlling the accessibility of the Shine-Dalgarno sequence, for example the expression of pathogenicity islands of some bacteria upon entry to a host. [3] [7] Recent data predict the existence of temperature-dependent alternative secondary structures (including Rho-independent terminators) upstream of cold shock proteins in E. coli. [3]

Discovery

Attenuation was first observed by Charles Yanofsky in the trp operon of E. coli . [8] The first observation was linked to two separate scientific facts. Mutations which knocked out the trp R (repressor) gene still showed some regulation of the trp operon (these mutants were not fully induced/repressed by tryptophan). The total range of trp operon regulation is about 700 X (on/off). When the trp repressor was knocked out, one still got about 10 X regulation by the absence or presence of trp. When the sequence of the beginning of the trp operon was determined an unusual open reading frame (ORF) was seen immediately preceding the ORFs for the known structural genes for the tryptophan biosynthetic enzymes. The general structural information shown below was observed from the sequence of the trp operon.

First, Yanofsky observed that the ORF contained two tandem Trp codons and the protein had a Trp percent composition which was about 10X normal. Second, the mRNA in this region contained regions of dyad symmetry which would allow it to form two mutually exclusive secondary structures. One of the structures looked exactly like a rho-independent transcription termination signal. The other secondary structure, if formed, would prevent the formation of this secondary structure and thus the terminator. This other structure is called the "preemptor".

The trp operon

Mechanism of transcriptional attenuation of the trp operon. Trp operon attenuation.svg
Mechanism of transcriptional attenuation of the trp operon.

An example is the trp gene in bacteria. When there is a high level of tryptophan in the region, it is inefficient for the bacterium to synthesize more. When the RNA polymerase binds and transcribes the trp gene, the ribosome will start translating. (This differs from eukaryotic cells, where RNA must exit the nucleus before translation starts.) The attenuator sequence, which is located between the mRNA leader sequence (5' UTR) and trp operon gene sequence, contains four domains, where domain 3 can pair with domain 2 or domain 4.

The attenuator sequence at domain 1 contains instruction for peptide synthesis that requires tryptophans. A high level of tryptophan will permit ribosomes to translate the attenuator sequence domains 1 and 2, allowing domains 3 and 4 to form a hairpin structure, which results in termination of transcription of the trp operon. Since the protein coding genes are not transcribed due to rho independent termination, no tryptophan is synthesised.

In contrast, a low level of tryptophan means that the ribosome will stall at domain 1, causing the domains 2 and 3 to form a different hairpin structure that does not signal termination of transcription. Therefore, the rest of the operon will be transcribed and translated, so that tryptophan can be produced. Thus, domain 4 is an attenuator. Without domain 4, translation can continue regardless of the level of tryptophan. [9] The attenuator sequence has its codons translated into a leader peptide, but is not part of the trp operon gene sequence. The attenuator allows more time for the attenuator sequence domains to form loop structures, but does not produce a protein that is used in later tryptophan synthesis.

Attenuation is a second mechanism of negative feedback in the trp operon. While the TrpR repressor decreases transcription by a factor of 70, attenuation can further decrease it by a factor of 10, thus allowing accumulated repression of about 700-fold. Attenuation is made possible by the fact that in prokaryotes (which have no nucleus), the ribosomes begin translating the mRNA while RNA polymerase is still transcribing the DNA sequence. This allows the process of translation to directly affect transcription of the operon.

At the beginning of the transcribed genes of the trp operon is a sequence of 140 nucleotides termed the leader transcript (trpL). This transcript includes four short sequences designated 1–4. Sequence 1 is partially complementary to sequence 2, which is partially complementary to sequence 3, which is partially complementary to sequence 4. Thus, three distinct secondary structures (hairpins) can form: 1–2, 2–3 or 3–4. The hybridization of strands 1 and 2 to form the 1–2 structure prevents the formation of the 2–3 structure, while the formation of 2-3 prevents the formation of 3–4. The 3–4 structure is a transcription termination sequence, once it forms RNA polymerase will disassociate from the DNA and transcription of the structural genes of the operon will not occur.

Part of the leader transcript codes for a short polypeptide of 14 amino acids, termed the leader peptide. This peptide contains two adjacent tryptophan residues, which is unusual, since tryptophan is a fairly uncommon amino acid (about one in a hundred residues in a typical E. coli protein is tryptophan). If the ribosome attempts to translate this peptide while tryptophan levels in the cell are low, it will stall at either of the two trp codons. While it is stalled, the ribosome physically shields sequence 1 of the transcript, thus preventing it from forming the 1-2 secondary structure. Sequence 2 is then free to hybridize with sequence 3 to form the 2-3 structure, which then prevents the formation of the 3-4 termination hairpin. RNA polymerase is free to continue transcribing the entire operon. If tryptophan levels in the cell are high, the ribosome will translate the entire leader peptide without interruption and will only stall during translation termination at the stop codon. At this point the ribosome physically shields both sequences 1 and 2. Sequences 3 and 4 are thus free to form the 3-4 structure which terminates transcription. The result is that the operon will be transcribed only when tryptophan is unavailable for the ribosome, while the trpL transcript is constitutively expressed.

To ensure that the ribosome binds and begins translation of the leader transcript immediately following its synthesis, a pause site exists in the trpL sequence. Upon reaching this site, RNA polymerase pauses transcription and apparently waits for translation to begin. This mechanism allows for synchronization of transcription and translation, a key element in attenuation.

A similar attenuation mechanism regulates the synthesis of histidine, phenylalanine and threonine.

Mechanism in the trp operon

The proposed mechanism of how this mRNA secondary structure and the trp leader peptide could regulate transcription of the trp biosynthetic enzymes includes the following.

The location of ribosomes determines which alternate secondary structures form.

Other operons controlled by attenuation

The discovery of this type of mechanism to control the expression of genes in a biosynthetic operon lead to its identification in a wide variety of such operons for which repressors had never been discovered. For example:

OperonLeader peptideArticle
HistidineMTRVQFKHHHHHHHPD stop Histidine operon leader
ThreonineMKRISTTITTTITITTGNGAG stop Threonine operon leader
Ilv (GEDA)MTALLRVISLVVISVVVIIIPPCGAALGRGKA stop
IlvBMTTSMLNAKLLPTAPSAAVVVVRVVVVVGNAP stop
LeucineMSHIVRFTGLLLLNAFIVRGRPVGGIQH stop Leucine operon leader/Lactis-leu-phe leader RNA motif
PhenylalanineMKHIPFFFAFFFTFP stop Lactis-leu-phe leader RNA motif

Attenuation in eukaryotes

Although an attenuation mechanism that involves translation while transcription is ongoing, like to the mechanism for the trp operon (and some other amino acid biosynthetic operons), would not work in eukaryotes, there is evidence for attenuation in Eukaryotes. [10] Research conducted on microRNA processing provides evidence of eukaryotic attenuation; after co-transcriptional endonucleolitical cleavage by Drosha 5'->3' exonuclease XRN2 may terminate further transcription by torpedo mechanism.

Related Research Articles

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

<span class="mw-page-title-main">RNA polymerase</span> Enzyme that synthesizes RNA from DNA

In molecular biology, RNA polymerase, or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template.

<span class="mw-page-title-main">Rho factor</span> Prokaryotic protein

A ρ factor is a bacterial protein involved in the termination of transcription. Rho factor binds to the transcription terminator pause site, an exposed region of single stranded RNA after the open reading frame at C-rich/G-poor sequences that lack obvious secondary structure.

In genetics, a transcription terminator is a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized transcript RNA that trigger processes which release the transcript RNA from the transcriptional complex. These processes include the direct interaction of the mRNA secondary structure with the complex and/or the indirect activities of recruited termination factors. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs.

<span class="mw-page-title-main">Tryptophan repressor</span> Transcription factor

Tryptophan repressor is a transcription factor involved in controlling amino acid metabolism. It has been best studied in Escherichia coli, where it is a dimeric protein that regulates transcription of the 5 genes in the tryptophan operon. When the amino acid tryptophan is plentiful in the cell, it binds to the protein, which causes a conformational change in the protein. The repressor complex then binds to its operator sequence in the genes it regulates, shutting off the genes.

In molecular biology, a termination factor is a protein that mediates the termination of RNA transcription by recognizing a transcription terminator and causing the release of the newly made mRNA. This is part of the process that regulates the transcription of RNA to preserve gene expression integrity and are present in both eukaryotes and prokaryotes, although the process in bacteria is more widely understood. The most extensively studied and detailed transcriptional termination factor is the Rho (ρ) protein of E. coli.

<span class="mw-page-title-main">Termination signal</span>

A termination signal is a sequence that signals the end of transcription or translation. Termination signals are found at the end of the part of the chromosome being transcribed during transcription of mRNA. Termination signals bring a stop to transcription, ensuring that only gene-encoding parts of the chromosome are transcribed. Transcription begins at the promoter when RNA polymerase, an enzyme that facilitates transcription of DNA into mRNA, binds to a promoter, unwinds the helical structure of the DNA, and uses the single-stranded DNA as a template to synthesize RNA. Once RNA polymerase reaches the termination signal, transcription is terminated. In bacteria, there are two main types of termination signals: intrinsic and factor-dependent terminators. In the context of translation, a termination signal is the stop codon on the mRNA that elicits the release of the growing peptide from the ribosome.

<i>trp</i> operon Operon that codes for the components for production of tryptophan

The trp operon is a group of genes that are transcribed together, encoding the enzymes that produce the amino acid tryptophan in bacteria. The trp operon was first characterized in Escherichia coli, and it has since been discovered in many other bacteria. The operon is regulated so that, when tryptophan is present in the environment, the genes for tryptophan synthesis are repressed.

<span class="mw-page-title-main">Amino acid synthesis</span> The set of biochemical processes by which amino acids are produced

Amino acid synthesis is the set of biochemical processes by which the amino acids are produced. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesize all amino acids. For example, humans can synthesize 11 of the 20 standard amino acids. These 11 are called the non-essential amino acids).

Antitermination is the prokaryotic cell's aid to fix premature termination of RNA synthesis during the transcription of RNA. It occurs when the RNA polymerase ignores the termination signal and continues elongating its transcript until a second signal is reached. Antitermination provides a mechanism whereby one or more genes at the end of an operon can be switched either on or off, depending on the polymerase either recognizing or not recognizing the termination signal.

<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

<span class="mw-page-title-main">Tryptophan operon leader</span>

The Tryptophan operon leader is an RNA element found at the 5′ of some bacterial tryptophan operons. The leader sequence can form two different structures known as the terminator and the anti-terminator, based on the Tryptophan amounts in the cell. The leader also codes for very short peptide sequence that is rich in tryptophan. The terminator structure is recognised as a termination signal for RNA polymerase and the operon is not transcribed. This structure forms when the cell has an excess of tryptophan and ribosome movement over the leader transcript is not impeded. When there is a deficiency of the charged tryptophanyl tRNA the ribosome translating the leader peptide stalls and the antiterminator structure can form. This allows RNA polymerase to transcribe the operon.

<span class="mw-page-title-main">Leucine operon leader</span>

The Leucine operon leader is an RNA element found upstream of the first gene in the Leucine biosynthetic operon. The leader sequence can assume two different secondary structures known as the terminator and the anti-terminator structure. The leader also codes for very short peptide sequence that is rich in leucine amino acid. The terminator structure is recognised as a termination signal for RNA polymerase and the operon is not transcribed. This structure forms when the cell has an excess of leucine and ribosome movement over the leader transcript is not impeded. When there is a deficiency of the charged leucyl tRNA the ribosome translating the leader peptide stalls and the antiterminator structure can form. This allows RNA polymerase to transcribe the operon. At least 6 amino acid operons are known to be regulated by attenuation.

<span class="mw-page-title-main">PreQ1 riboswitch</span>

The PreQ1-I riboswitch is a cis-acting element identified in bacteria which regulates expression of genes involved in biosynthesis of the nucleoside queuosine (Q) from GTP. PreQ1 (pre-queuosine1) is an intermediate in the queuosine pathway, and preQ1 riboswitch, as a type of riboswitch, is an RNA element that binds preQ1. The preQ1 riboswitch is distinguished by its unusually small aptamer, compared to other riboswitches. Its atomic-resolution three-dimensional structure has been determined, with the PDB ID 2L1V.

<span class="mw-page-title-main">Histidine operon leader</span>

The Histidine operon leader is an RNA element found in the bacterial histidine operon. At least 6 amino acid operons are known to be regulated by attenuation. In each a leader sequence of 150–200 bp is found upstream of the first gene in the operon. This leader sequence can assume two different secondary structures known as the terminator and the anti-terminator structure. In each case the leader also codes for very short peptide sequence that is rich in the end product amino acid of the operon. The terminator structure is recognised as a termination signal for RNA polymerase and the operon is not transcribed. This structure forms when the cell has an excess of the regulatory amino acid and ribosome movement over the leader transcript is not impeded. When there is a deficiency of the charged tRNA of the regulatory amino acid the ribosome translating the leader peptide stalls and the antiterminator structure can form. This allows RNA polymerase to transcribe the operon.

<span class="mw-page-title-main">Threonine operon leader</span>

The threonine operon leader is an RNA element. Threonine is one of at least 6 amino acid operons are known to be regulated by attenuation. In each a leader sequence of 150–200 bp is found upstream of the first gene in the operon. This leader sequence can assume two different secondary structures known as the terminator and the anti-terminator structure. In each case the leader also codes for very short peptide sequence that is rich in the end product amino acid of the operon. The terminator structure is recognised as a termination signal for RNA polymerase and the operon is not transcribed. This structure forms when the cell has an excess of the regulatory amino acid and ribosome movement over the leader transcript is not impeded. When there is a deficiency of the charged tRNA of the regulatory amino acid the ribosome translating the leader peptide stalls and the antiterminator structure can form. This allows RNA polymerase to transcribe the operon.

<span class="mw-page-title-main">Intrinsic termination</span>

Intrinsic, or rho-independent termination, is a process in prokaryotes to signal the end of transcription and release the newly constructed RNA molecule. In prokaryotes such as E. coli, transcription is terminated either by a rho-dependent process or rho-independent process. In the Rho-dependent process, the rho-protein locates and binds the signal sequence in the mRNA and signals for cleavage. Contrarily, intrinsic termination does not require a special protein to signal for termination and is controlled by the specific sequences of RNA. When the termination process begins, the transcribed mRNA forms a stable secondary structure hairpin loop, also known as a Stem-loop. This RNA hairpin is followed by multiple uracil nucleotides. The bonds between uracil and adenine are very weak. A protein bound to RNA polymerase (nusA) binds to the stem-loop structure tightly enough to cause the polymerase to temporarily stall. This pausing of the polymerase coincides with transcription of the poly-uracil sequence. The weak adenine-uracil bonds lower the energy of destabilization for the RNA-DNA duplex, allowing it to unwind and dissociate from the RNA polymerase. Overall, the modified RNA structure is what terminates transcription.

Post-transcriptional regulation is the control of gene expression at the RNA level. It occurs once the RNA polymerase has been attached to the gene's promoter and is synthesizing the nucleotide sequence. Therefore, as the name indicates, it occurs between the transcription phase and the translation phase of gene expression. These controls are critical for the regulation of many genes across human tissues. It also plays a big role in cell physiology, being implicated in pathologies such as cancer and neurodegenerative diseases.

<span class="mw-page-title-main">Archaeal transcription</span>

Archaeal transcription is the process in which a segment of archaeal DNA is copied into a newly synthesized strand of RNA using the sole Pol II-like RNA polymerase (RNAP). The process occurs in three main steps: initiation, elongation, and termination; and the end result is a strand of RNA that is complementary to a single strand of DNA. A number of transcription factors govern this process with homologs in both bacteria and eukaryotes, with the core machinery more similar to eukaryotic transcription.

Transcription-translation coupling is a mechanism of gene expression regulation in which synthesis of an mRNA (transcription) is affected by its concurrent decoding (translation). In prokaryotes, mRNAs are translated while they are transcribed. This allows communication between RNA polymerase, the multisubunit enzyme that catalyzes transcription, and the ribosome, which catalyzes translation. Coupling involves both direct physical interactions between RNA polymerase and the ribosome, as well as ribosome-induced changes to the structure and accessibility of the intervening mRNA that affect transcription.

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