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miRNA BINDS TARGET RNA

FIGURE 11.14 Micro RNA

Micro RNA (miRNA) is made by processing a longer precursor that folds into a stem and loop. Dicer, the same nuclease that makes siRNA, is responsible for this processing. After strand separation, one strand of the miRNA binds to the target mRNA and prevents translation.

miRNA

HTTTTTTT rTTTTTT

Translation is blocked mRNA

Micro RNA is a kind of short regulatory RNA that blocks translation of mRNA.

Micro RNA molecules are produced from longer RNA precursors of approximately 70 nucleotides that are transcribed from chromosomal genes. These precursor RNA molecules fold into stem-loop structures (Fig. 11.14). The double-stranded stem region is then cut by Dicer, the same nuclease that generates siRNA. The miRNA molecules have 1-3 unpaired bases in the middle and thus differ from siRNA, which is completely base paired. Instead of Rde1, which recognizes siRNA, the miRNA is bound by the related proteins, Alg1 and Alg2 (in C. elegans). It is uncertain whether the RISC complex then binds to the miRNA and separates the strands (as for siRNA). An alternative is that the strands separate spontaneously since miRNA is imperfectly base-paired. In any case, one strand of the miRNA then binds to the target mRNA. However, base pairing is not usually perfect (as it would be with siRNA). The result

Premature Termination Causes Attenuation of RNA Transcription 297

Premature Termination Causes Attenuation of RNA Transcription 297

FIGURE 11.15 Alternative Secondary Structures of mRNA Leader Region

The sequences in the leader region of the messenger RNA designated by 1, 2, 3 and 4 can base pair in two alternative ways. A) The structure for premature termination of mRNA is due to base pairing that forms two stem and loop structures in the mRNA. The second loop, 3 plus 4, causes termination. B) Termination can be prevented if a protein binds to the mRNA at site 1, allowing sites 2 and 3 to pair off. This creates the "pre-emptor" and prevents the terminator from forming.

FIGURE 11.15 Alternative Secondary Structures of mRNA Leader Region

The sequences in the leader region of the messenger RNA designated by 1, 2, 3 and 4 can base pair in two alternative ways. A) The structure for premature termination of mRNA is due to base pairing that forms two stem and loop structures in the mRNA. The second loop, 3 plus 4, causes termination. B) Termination can be prevented if a protein binds to the mRNA at site 1, allowing sites 2 and 3 to pair off. This creates the "pre-emptor" and prevents the terminator from forming.

is that translation of the mRNA is blocked, but the mRNA is not degraded. The precise mechanism of inhibition of translation is presently uncertain.

Micro RNA is found in worms, insects, mammals and plants—i.e. the same organisms that display RNA interference. The first miRNAs were found in C. elegans where they were called small temporal RNA (stRNA) because they regulate the timing of worm development during the conversion of the larva into the adult. Many miRNAs appear to be involved in the regulation of development and many of the targets for miRNA are mRNAs that encode transcription factors, which in turn regulate the expression of other genes.

Regulation by attenuation involves alternative stem and loop structures in the mRNA.

Premature Termination Causes Attenuation of RNA Transcription

Transcriptional attenuation is a regulatory mechanism that involves premature termination of mRNA synthesis. The basic principle of attenuation is that the first part of the mRNA to be made, the leader region, can fold up into two alternative secondary structures. One of these allows continued transcription but the other secondary structure causes premature termination. The status of attenuation is somewhat ambiguous. It is often viewed as regulation at the level of transcription. However, it does involve mRNA that has already been partly transcribed. Consequently it is sometimes regarded as "post-transcriptional". Since attenuation is closely related to other mechanisms based on alternative RNA stem and loop structures, I have chosen to include it along with other forms of RNA based regulation.

Typically, the leader region contains four sub-regions (sequences 1 through 4) that may base pair in two different ways. When no other factors intervene, sequence 1 pairs with 2 and sequence 3 pairs with 4, so forming two stem and loop structures (Fig. 11.15). The second of these stem and loop structures, containing paired sequences 3 and 4, acts as a terminator. However, sequence 2 may instead pair with 3. For this to happen, a protein must bind to sequence 1 and remove it from play. The net result is that the terminator loop, normally consisting of sequences 3 and 4, no longer forms and transcription of the mRNA can continue.

attenuation Type of transcriptional regulation that works by premature termination and depends on alternative stem and loop structures in the leader region of the mRNA

leader region The region of an mRNA molecule in front of the structural genes, especially when involved in regulation by the attenuation mechanism

A. Sequence layout

mRNA

Coding sequence for leader peptide

B. Gene on

(No critical amino acids)

C. Gene off

(Critical amino acid present)

B. Gene on

(No critical amino acids)

FIGURE 11.16 Stalled Ribosome Prevents Formation of Terminator Loop

5' end mRNA

Leader peptide made of amino acids

FIGURE 11.16 Stalled Ribosome Prevents Formation of Terminator Loop

Attenuation controls whether synthesis of mRNA is completed or aborted. [The RNA polymerase is not shown in this figure, just the attenuation mechanism.] (A) The leader region of the mRNA contains the coding sequence for the leader peptide and four specific sequences (1, 2, 3 and 4) that can base pair to form stem and loop structures. (B) When there is a shortage of the corresponding amino acid, the ribosome slows down at region 1, allowing the pre-emptor structure to form and transcription of the mRNA by RNA poly merase to continue. Note that the leader peptide is not com pleted. (C) When there is an abundance of the corresponding amino acid, the leader peptide is made and the ribosome quickly moves to region 2, allowing regions 3 and 4 to form the terminator loop. This prevents further elongation of the mRNA.

Attenuation is used to regulate the genes for biosynthesis of amino acids in both gram-negative bacteria, such as E. coli, and gram-positive bacteria, such as Bacillus. If the supply of amino acid is plentiful, then the genes for its biosynthesis should be turned off. Conversely, if the level of the amino acid is low, the biosynthetic genes should be transcribed.

In E. coli, attenuation is complicated and usually involves the binding of ribosomes to the mRNA leader region where they translate a leader peptide. The leader peptide is encoded by a short open reading frame and only consists of 14 or 15 amino acids. It lies close to the 5'-end of the mRNA, upstream of the structural genes for the enzymes of the biosynthetic pathway (Fig. 11.16A).

The leader peptide contains several tandem codons for the amino acid in question. For example, in the leader peptide of the his operon of E. coli there are seven codons for histidine in a row. In the leader peptide of the thr operon, there are 11 clustered codons for threonine and isoleucine. Since threonine is the precursor for isoleucine, codons for both of these amino acids are included in attenuation control. When an amino acid is in short supply, the ribosome has difficulty finding a charged tRNA carrying that particular amino acid and it slows down. When several codons for a scarce amino acid follow each other, the ribosome grinds to a halt. The stalled ribosome covers sequence 1, loop 2/3 forms and the terminator loop is not made (Fig. 11.16B). The RNA polymerase carries on, transcribing the rest of the mRNA.

In Bacillus, ribosomes are not involved and there is no leader peptide. Nonetheless, the leader region of the mRNA possesses four sequences that can pair up

FIGURE 11.17 Attenuation by RNA-Binding Protein

When tryptophan is present, it binds avidly to the tryptophan attenuation protein. This binds to the RNA and alters its structure. The alternative RNA structure possesses a stem and loop that causes premature termination.

to give two alternative structures. An attenuation protein binds the amino acid in question. In the presence of the amino acid, the attenuation protein binds to the mRNA leader region and promotes termination. In Bacillus, the 5'-region of the leader of trp mRNA contains a run of eleven UAG or GAG triplets separated from each other by two or three other bases. Eleven subunits of the tryptophan attenuation protein (TRAP) bind to these, forming an eleven-membered ring (Fig. 11.17). This allows formation of the terminator stem and loop and so causes premature termination of transcription.

Riboswitches—RNA Acting Directly as a Control Mechanism

One of the most fascinating recent stories in molecular biology has been the discovery that RNA can carry out many of the functions that were previously believed to need proteins. Ribozymes, that is to say catalytically active RNA, are involved in RNA splicing, protein synthesis and viroid replication. Antisense RNA and a variety of small regulatory RNA molecules, such as siRNA are involved in gene regulation. Most recently, it has been found that RNA domains at the front of messenger RNA, referred to as riboswitches, can directly interact with small molecules and can control gene expression. The vast majority of riboswitches have been found in bacteria and so far it is only in bacteria that experimental evidence for riboswitch operation exists.

A few messenger RNA molecules can control their own translation via riboswitch domains that bind small molecules.

attenuation protein Regulatory protein involved in attenuation and that binds to the leader region of mRNA

riboswitch Domain of messenger RNA that directly senses a signal and controls translation by alternating between two structures

FIGURE 11.18 Riboswitch Mechanisms

Riboswitches alternate between two alternative stem and loop structures depending on the presence or absence of the signal metabolite. (A) In the attenuation mechanism, the presence of the signal metabolite results in formation of the terminator structure and transcription is aborted. (B) In the translational inhibition mechanism, the presence of the metabolite results in sequestration of the Shine-Dalgarno sequence, which prevents translation of the mRNA.

FIGURE 11.18 Riboswitch Mechanisms

Riboswitches alternate between two alternative stem and loop structures depending on the presence or absence of the signal metabolite. (A) In the attenuation mechanism, the presence of the signal metabolite results in formation of the terminator structure and transcription is aborted. (B) In the translational inhibition mechanism, the presence of the metabolite results in sequestration of the Shine-Dalgarno sequence, which prevents translation of the mRNA.

However, sequence analysis has revealed that the genomes of certain fungi and plants contain equivalent sequences, implying that they probably have riboswitches too.

Biosynthetic pathways that make metabolites such as amino acids and vitamins are generally induced when the metabolite is in short supply but are shut down when there is a plentiful supply of the metabolite. The genes for such pathways are often controlled by repressors or by attenuation and are repressed in response to high concentrations of the metabolite in question. In these cases, the metabolite is bound by a regulatory protein as already described, such as the ArgR repressor of E. coli which binds arginine, or the tryptophan attenuation protein (TRAP) of Bacillus that binds tryptophan.

In riboswitches, the metabolite is directly bound by an RNA sequence at the 5'-end of the messenger RNA. For example, the thiamine riboswitch of E. coli contains a sequence that binds the vitamin/cofactor thiamine pyrophosphate with great specificity and is known as the THI box. Similarly the RFN box of the riboflavin riboswitch in Bacillus subtilis binds flavin mononucleotide. When these vitamins are in short supply the biosynthetic genes are turned on without the intervention of any regulatory protein. Conversely, when the vitamin is present at high levels the genes are turned off. Riboswitches are presently known for several vitamins, a few amino acids (methio-nine and lysine) and the purine bases adenine and guanine.

Binding of the metabolite to its RNA box changes the conformation of the whole riboswitch domain. Riboswitches exist in two alternative conformations that have different stem and loop structures. This in turn controls gene expression by one of two related mechanisms, premature termination of transcription (i.e. attenuation) or trans-lational inhibition:

In the attenuation mechanism (Fig 11.18A), the riboswitch controls whether or not the mRNA for the biosynthetic genes will be prematurely terminated. Here the riboswitch sequesters the terminator sequences in the absence of the signal metabolite and transcription continues. When the metabolite binds, the riboswitch changes to a conformation that allows the formation of a terminator stem and loop, which causes premature termination of the mRNA. Consequently, the genes are not expressed.

In the translational inhibition mechanism (Fig 11.18B), the riboswitch controls whether or not the mRNA will be translated. When the signal metabolite is absent the Shine-Dalgarno sequence is free to bind to ribosomal RNA and translation proceeds. When the signal metabolite binds, the riboswitch sequesters the Shine-Dalgarno sequence and translation is prevented.

Riboswitches can respond to temperature as well as to metabolite concentrations.

Riboswitches can also respond to physical conditions as opposed to small molecules. The RNA thermosoensor is a specialized kind of riboswitch that responds to temperature and controls mRNA translation by sequestration of the Shine-Dalgarno sequence. The principle is the same as above, but formation of the alternative stem and loop structures depends directly on temperature. At high temperature one of the stems is unstable and the riboswitch flips to its high temperature form. The rpoH gene of E. coli is involved in the heat shock response, as described in Chapter 9. In addition to the regulation described there, translation of rpoH mRNA is prevented by an RNA thermosoensor at low temperature but allowed as the temperature increases. Other genes whose translation is controlled by RNA thermosoensors include certain activator genes involved in the virulence of pathogenic bacteria such as Yersinia and Listeria. Outside the host, these genes are switched off by translational inhibition. Inside the hot-blooded mammalian host the temperature is warmer and the activator proteins are expressed. The activator proteins then proceed to activate several other genes involved in bacterial virulence.

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