Chapter 13 Regulatory RNA

13.1 Introduction RNA functions as a regulator by forming a region of secondary structure (either inter- or intramolecular) that changes the properties of a target sequence. Key Concepts

The basic principle of regulation in bacteria is that gene expression is controlled by a regulator that interacts with a specific sequence or structure in DNA or mRNA at some stage prior to the synthesis of protein. The stage of expression that is controlled can be transcription, when the target for regulation is DNA, or it can be at translation, when the target for regulation is RNA. When control is during transcription, it can be at initiation or at termination. The regulator can be a protein or an RNA. Regulators may themselves be regulated, most typically in response to small molecules whose supply responds to environmental condition. Regulators may be controlled by other regulators to make complex circuits.

Regulation via RNA; intramolecular or intermolecular interaction. The most common role for intramolecular changes is for an RNA molecule to assume alternative secondary structures by utilizing different schemes for base pairing. In intermolecular interactions, an RNA regulator recognizes its target by the familiar principle of complementary base pairing. Fig. 13.1: shows that the regulator is usually a small RNA molecule with extensive secondary structure, but with a single-stranded region(s) that is complementary to a single-stranded region in its target.

Figure A regulator RNA is a small RNA with a single- stranded region that can pair with a single-stranded region in a target RNA.

13.2 Alternative Secondary Structures Control Attenuation Termination of transcription can be attenuated by controlling formation of the necessary hairpin structure in RNA. The most direct mechanisms for attenuation involve proteins that either stabilize or destabilize the hairpin. Key Concepts

RNA structure provides an opportunity for regulation in both prokaryotes and eukaryotes. Its most common role occurs when an RNA molecule can take up alternative secondary structure by utilizing different schemes for intramolecular base pairing. This type of mechanism can be used to regulate the termination of transcription, when the alternative structures differ in whether they permit termination. Another means of controlling conformation is provided by the cleavage of an RNA; by removing one segment of an RNA, the conformation of the rest may be altered. -Attenuation: describes the regulation of bacterial operons by controlling termination of transcription at a site located before the first structural gene. -attenuator: is a terminator sequence at which attenuation occurs.

The principle of attenuation is that some external event controls the formation of the hairpin needed for intrinsic termination. If the hairpin is allowed to form, termination prevents RNA polymerase from transcribing the structural genes. If the hairpin is prevented from forming, RNA polymerase elongates through the terminator and the genes are expressed. Fig. 13.2: shows an example in which a protein prevents formation of the terminator hairpin.

Figure Attenuation occurs when a terminator hairpin in RNA is prevented from forming.

13.3 Termination of B. subtilis trp Genes Is Controlled by Tryptophan and by tRNA Trp A terminator protein called TRAP is activated by tryptophan to prevent transcription of trp genes. Activity of TRAP is (indirectly) inhibited by uncharged tRNA Trp. Key Concepts

The circuitry that controls transcription via termination can use both direct and indirect means to respond to the level of small molecule products or substrates. In B. subtilis, a protein called tryptophan RNA-binding attenuation protein (TRAP) is activated by tryptophan (Trp) to bind to a sequence in the leader of the nascent transcript. TRAP forms a multimer of eleven subunits. Each subunit binds a single tryptophan amino acid and a trinucleotide (GAG or UAG) of RNA. The RNA is wound in a circle around the protein. Fig. 13.3: shows that the result is to ensure the availability of the regions that are required to form the terminator hairpin.

Figure TRAP is activated by tryptophan and binds to trp mRNA. This allows the termination hairpin to form, with the result that RNA polymerase terminates, and the genes are not expressed. In the absence of tryptophan, TRAP does not bind, and the mRNA adopts a structure that prevents the terminator hairpin from forming.

The TRAP protein in turn, however, is also controlled by tRNA Trp. Fig. 13.4: shows that uncharged tRNA Trp binds to the mRNA for a protein called antiTRAP (AT). This suppresses formation of a termination hairpin in the mRNA. Expression of the B. subtilis trp genes is therefore controlled by both tryptophan and tRNA Trp.

Figure Under normal conditions (in the presence of tryptophan) transcription terminates before the anti-TRAP gene. When tryptophan is absent, uncharged tRNA Trp base pairs with the anti- TRAP mRNA, preventing formation of the terminator hairpin, thus causing expression of anti-TRAP.

13.4 The E. coli tryptophan Operon Is Controlled by Attenuation An attenuator (intrinsic terminator) is located between the promoter and the first gene of the trp cluster. The absence of tryptophan suppresses termination and results in a 10× increase in transcription. Key Concepts

A complex regulatory system is used in E. coli (where attenuation was originally discovered). The changes in secondary structure that control attenuation are determined by the position of the ribosome on mRNA. Fig. 13.5: shows that termination requires that the ribosome can translate a leader segment that precedes that trp genes in the mRNA. When the ribosome translates the leader region, a termination hairpin forms at terminator 1. When the ribosome is prevented from translating the leader, though, the termination hairpin does not form, and RNA polymerase transcribes the coding region. This mechanism of antitermination therefore depends upon the ability of external circumstances to influence ribosome movement in the leader region.

Figure Termination can be controlled via changes in RNA secondary structure that are determined by ribosome movement.

The trp operon consists of 5 structural genes, which code for the 3 enzymes that convert chorismic acid to tryptophan. Fig. 13.6: shows that transcription starts at a promoter at the left end of the cluster. Trp operon expression is controlled by two separate mechanisms; a repressor protein (coded by the unlinked gene trpR) that binds to an operator, and attenuation. An attenuator (intrinsic terminator) is located between the promoter and the trpE gene. RNA polymerase terminates there to produce a 140-base transcript. Fig. 13.7: termination at the attenuator responds to the level of tryptophan.

Figure The trp operon consists of five contiguous structural genes preceded by a control region that includes a promoter, operator, leader peptide coding region, and attenuator.

Figure An attenuator controls the progression of RNA polymerase into the trp genes. RNA polymerase initiates at the promoter and then proceeds to position 90, where it pauses before proceeding to the attenuator at position 140. In the absence of tryptophan, the polymerase continues into the structural genes (trpE starts at +163). In the presence of tryptophan there is ~90% probability of termination to release the 140-base leader RNA.

13.5 Attenuation Can Be Controlled by Translation The leader region of the trp operon has a fourteen-codon open reading frame that includes two codons for tryptophan. The structure of RNA at the attenuator depends on whether this reading frame is translated. In the presence of tryptophan, the leader is translated, and the attenuator is able to form the hairpin that causes termination. In the absence of tryptophan, the ribosome stalls at the tryptophan codons and an alternative secondary structure prevents formation of the hairpin, so that transcription continues. Key Concepts

How can termination of transcription at the attenuator respond to the level of tryptophan? The leader region has a short coding sequence that could represent a leader peptide of 14 amino acids. Fig. 13.6: shows that it contains a ribosome binding site whose AUG codon is followed by a short coding region that contains two successive codons for tryptophan. When the cell runs out of tryptophan, ribosomes initiate translation of the leader peptide but stop when they reach the Trp codons. This ribosome stalling influences termination at the attenuator.. -Leader peptide: is the product that would result from translation of a short coding sequence used to regulate expression of the tryptophan by controlling -Ribosome stalling: describes the inhibition of movement that occurs when a ribosome reaches a codon for which there is no corresponding charged aminoacyl-tRNA.

Fig. 13.8: The leader sequence can be written in alternative base- paired structure (Left: terminator hairpin). Fig. 13.9: shows that the position of the ribosome can determine which structure is formed in such a way that termination is attenuated only in the absence of tryptophan. Control by attenuation requires a precise timing of events. Translation of the leader must occur at the same time when RNA polymerase approaches the terminator site. Fig : summarizes the role of Trp-tRNA in controlling expression of the operon.

Figure The trp leader region can exist in alternative base-paired conformations. The center shows the four regions that can base pair. Region 1 is complementary to region 2, which is complementary to region 3, which is complementary to region 4. On the left is the conformation produced when region 1 pairs with region 2, and region 3 pairs with region 4. On the right is the conformation when region 2 pairs with region 3, leaving regions 1 and 4 unpaired.

Figure The alternatives for RNA polymerase at the attenuator depend on the location of the ribosome, which determines whether regions 3 and 4 can pair to form the terminator hairpin.

Figure In the presence of tryptophan tRNA, ribosomes translate the leader peptide and are released. This allows hairpin formation, so that RNA polymerase terminates. In the absence of tryptophan tRNA, the ribosome is blocked, the termination hairpin cannot form, and RNA polymerase continues.

13.6 Antisense RNA Can Be Used to Inactivate Gene Expression Antisense genes block expression of their targets when introduced into eukaryotic cells. Key Concepts There are many cases in both prokaryotes and eukaryotes where a (usually rather short) single-stranded RNA base pairs with a complementary region of an RNA, and as a result it prevents expression of the mRNA. -antisense gene: codes for an (antisense) RNA that has a complementary sequence to an RNA that is its target. Fig : antisense genes are constructed by reversing the orientation of a gene with regard to its promoter, so that the “antisense” strand is transcribed. An antisense RNA is in effect a synthetic RNA regulator.

Figure Antisense RNA can be generated by reversing the orientation of a gene with respect to its promoter, and can anneal with the wild-type transcript to form duplex RNA.

13.7 Small RNA Molecules Can Regulate Translation A regulator RNA functions by forming a duplex region with a target RNA. The duplex may block initiation of translation, cause termination of transcription, or create a target for an endonuclease. Key Concepts

Like a protein regulator, a small regulator RNA is an independently synthesized molecule that diffuses to a target site consisting of a specific nucleotide sequence. The regulator RNA functions by complementarity with its target, at which it can form a double-stranded region. Two general mechanisms for the action of a regulator RNA: (1) Formation of a duplex region with the target nucleic acid directly prevents its ability to function by forming or sequestering a specific site. (Fig ; Fig ); (2) Formation of a duplex region in one part of the target molecule changes the conformation of another region, thus indirectly affecting its function (Fig ) Fig : riboswitch (ribozyme) provides an exception. Another regulatory mechanism that involves transcription of a noncoding RNA works indirectly. Initiation at a target promoter can be suppressed by transcription from another promoter upstream from it (Fig )

Figure A protein that binds to a single-stranded region in a target RNA could be excluded by a regulator RNA that forms a duplex in this region.

Figure By binding to a target RNA to form a duplex region, a regulator RNA may create a site that is attacked by a nuclease.

Figure The secondary structure formed by base pairing between two regions of the target RNA may be prevented from forming by base pairing with a regulator RNA. In this example, the ability of the 3′ end of the RNA to pair with the 5′ end is prevented by the regulator.

Figure The 5’ untranslated region of the mRNA for the enzyme that synthesizes GlcN6P (glucosamine-6-phosphate) contains a ribozyme that is activated by the metabolic product. The ribozyme inactivates the mRNA by cleaving it.

Figure Transcription from an upstream promoter may inhibit initiation at a promoter downstream from it, because reading through the downstream promoter prevents the necessary transcription factors from binding to it. The RNA transcribed from the upstream promoter has no coding function.

13.8 Bacteria Contain Regulator RNAs Bacterial regulator RNAs are called sRNAs. Several of the sRNAs are bound by the protein Hfq, which increases their effectiveness. The OxyS sRNA activates or represses expression of >10 loci at the post-transcriptional level. Key Concepts

In bacteria, regulator RNAs are short molecules that are collectively known as sRNAs; E. coli contains at least 17 different sRNAs. Some of the sRNAs are general regulators that affect many target genes. -sRNA: is a small bacterial RNA that functions as a regulator of gene expression. Oxidative stress provides an interesting example of a general control system in which RNA is the regulator. When exposed to reactive oxygen species, bacteria respond by inducing antioxidant defense genes. Hydrogen peroxide activates the transcription activator OxyR which controls the expression of several inducible genes, One of these genes is oxyS, which codes for a small RNA (Fig ).

The OxyS RNA is a short sequence (109 nt) that does not code for protein. It is a trans-acting regulator that affects gene expression at posttranscriptional levels. It has >10 target loci; at some of them, it activates expression; at others it represses expression. Fig shows the mechanism of repression of one target, the FlhA mRNA.

Figure The gels on the left show that oxyS RNA is induced in an oxyR constitutive mutant. The gels on the right show that oxyS RNA is induced within 1 minute of adding hydrogen peroxide to a wild-type culture.

Figure oxyS RNA inhibits translation of flhA mRNA by base pairing with a sequence just upstream of the AUG initiation codon.

13.9 MicroRNAs Are Regulators in Many Eukaryotes Animal and plant genomes code for many short (~22 base) RNA molecules, called microRNAs. MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs. Key Concepts

Very small RNAs are gene regulators in many eukaryotes. The first example was discovered in the nematode C. elegans as the result of the interaction between the regulator gene lin4 and its target gene, lin14 (Fig ). The lin4 RNA is an example of a microRNA (miRNA). There are ~80 genes in the C. elegans genome coding for miRNAs that are 21 to 24 nucleotides long. -MicroRNAs are very short RNAs that may regulate gene expression.

Figure lin4 RNA regulates expression of lin14 by binding to the 3′ nontranslated region.

Many of the C. elegans miRNAs have homologs in mammals, so the mechanism may be widespread. They are also found in plants. The mechanism of production of the miRNAs is also widely conserved. In the example of lin4, the gene is transcribed into a transcript that forms a double-stranded region that becomes a target for a nuclease called Dicer. This has an N-terminal helicase activity, which enables it to unwind the double-stranded region, and two nuclease domains that are related to the bacterial ribonuclease III. Cleavage of the initial transcript generates the active miRNA.

13.10 RNA Interference Is Related to Gene Silencing RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA. dsRNA may cause silencing of host genes. Key Concepts

The regulation of mRNAs by miRNAs is mimicked by the phenomenon of RNA interference (RNAi). -RNA interference (RNAi): describes situations in which antisense and sense RNAs apparently are equally effective in inhibiting expression of a target gene. It is caused by the ability of double- stranded sequences to cause degradation of sequences that are complementary to them. The dsRNA is degraded by ATP-dependent cleavage to give oligonuclotides of 21 to 23 bases. The short RNA is sometimes called siRNA (short interfering RNA).

Fig : shows that the mechanism of cleavage involves making breaks relative to each 3’ end of a long dsRNA to generate siRNA fragments with short (two base) protruding 3’ ends. The same enzyme (Dicer) that generates miRNAs is responsible for the cleavage. RNAi occurs posttranscriptionally when an siRNA induces degradation of a complementary mRNA (Fig ). The siRNA directs cleavage of the mRNA in the middle of the paired segment. These reactions occur within a ribonucleoprotein complex called RISC (RNA-induced silencing complex). Fig : dsRNA has both general and specific effects. -RNA silencing: describes the ability of a dsRNA to suppress expression of the corresponding gene systemically in a plant. -Cosuppression: describes the ability of a transgene (usually in plants) to inhibit expression of the corresponding endogenous gene.

Figure siRNA that mediates RNA interference is generated by cleaving dsRNA into smaller fragments. The cleavage reaction occurs nucleotides from a 3′ end. The siRNA product has protruding bases on its 3′ ends.

Figure RNAi occurs when a dsRNA is cleaved into fragments that direct cleavage of the corresponding mRNA.

Figure dsRNA inhibits protein synthesis and triggers degradation of all mRNA in mammalian cells as well as having sequence-specific effects.

General mechanism of RNA-induced gene silencing Dicer Complex AAAA RDRP siRNAs miRNAs AGO AAAA RISC effector complex DNA/Histone Methylation mRNA Degradation Translation Block TGS RNAi/PTGS/VIGS Developmental Processes dsRNA