BIOCHEMISTRY DISCUSSION 9

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So now on to RNA synthesis, which is the second step in the central dogma of molecular biology. And once again we're going to start with bacteria, because they're a little bit more simple. And we'll be able to study certain processes that are common with all life forms. But we'll get into some of the more complicated processes that’re specific to the eukaryotic cells in the next chapter.

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Biochemistry: A Short Course

Fourth Edition

CHAPTER 36 RNA Synthesis and

Regulation in Bacteria

Tymoczko • Berg • Gatto • Stryer

So we'll start out by talking about RNA polymerase. Just like DNA polymerase, this is what's responsible for synthesizing the RNA in the cell. There's three stages of RNA synthesis: there’s initiation, elongation, and termination. And then finally we'll talk about the lac operon, which is one of the classic paradigms in genetic regulatory control.

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Chapter 36: Outline

36.1 Cellular RNA Is Synthesized by RNA Polymerases 36.2 RNA Synthesis Comprises Three Stages 36.3 The lac Operon Illustrates the Control of Bacterial

Gene Expression

So I'd like for you to watch this video, which will show you the overall picture of the transcription of RNA and the translation of that RNA into protein. Now, this video is specifically for eukaryotic cells, and includes a section on RNA editing which will not be covered in the bacterial section, but will be covered later. And I also wanted you to look at how the protein is folding as it’s being synthesized because I think that that's a pretty good representation of how it might happen.

Figure 36.1 RNA polymerase. This large enzyme comprises many subunits, including b (blue) and b¢ (red), which form a “claw” that holds the DNA to be transcribed. Notice that the active site includes a magnesium ion (green) at the center of the structure. [Drawn from IL9Z.pdb.]

So here's a representation of the bacterial RNA polymerase. And the DNA fits in that sort of cleft in the side, on the right hand side - looks like a claw, it sort of closes around.

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Structure of Bacterial RNA Polymerase

Here is a representation of yeast RNA polymerase. Yeast is a eukaryotic cell, so it's a little bit different from the bacterial cell, but I thought that this was a kind of instructive picture that makes you think about like what it might look like.

Figure 36.2 Complementarity between mRNA and DNA. The base sequence of mRNA (green) is the complement of that of the DNA template strand. The other strand of DNA is called the coding strand because it has the same sequence as that of the RNA transcript except for thymine (T) in place of uracil (U).

So RNA synthesis is done from a DNA template. And it's called transcription - and that kind of makes sense because you're basically rewriting the sequence that you find in the DNA - and that's catalyzed by an RNA polymerase. Now, RNA polymerase is similar to DNA polymerase - it requires a template, and the template in this case is the DNA. And the RNA is synthesized complementary to the DNA template.

So If you’ll take a look at the bottom, you'll see that there's two strands of DNA and then a strand of RNA in green. RNA, just like DNA, is synthesized in the 5' to 3' direction, so it's synthesized on the strand of the DNA that's going from 3' to 5'. That would be the complementary sequence. But you can see that the sequence of RNA is actually very analogous to the coding strand which is the other 5' to 3' DNA strand which is also complementary to the template. The RNA has U instead of T (that’s uracil instead of thymine), and when we write down the sequence of a gene, we usually write down the coding strand because that's the strand that is actually more representative of what the mRNA will look like. The template strand can always be inferred from the coding strand because they're complementary.

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Section 36.1 Cellular RNA Is Synthesized by RNA Polymerases (1/2)

Learning objective 1: Identify the key enzyme required for transcription. • The synthesis of RNA from a DNA template is called transcription, a process

catalyzed by RNA polymerase. RNA polymerase has the following requirements:

1. A template. The sequence of the newly synthesized RNA is complementary to the DNA template. The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the coding strand.

2. Activated precursors in the form of the four ribonucleoside triphosphates.

3. Divalent metal ions, usually Mg2+ or Mn2+.

Of course, you also need the activated ribonucleoside triphosphates, and some divalent metal ions in the active site for the actual catalysis of the synthesis.

Figure 36.3 RNA strand-elongation reaction.

The synthesis of RNA proceeds almost exactly like the synthesis of DNA. The 3' oxygen on the ribose attacks the α-phosphate group of an incoming nucleotide, and there're two phosphates that leave, and a phosphodiester bond is formed, and that is how the strand is grown. Complementary nucleotides come in, and very similar to DNA, they have to fit properly in order to be enclosed properly in the active site and have this process occur.

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Section 36.1 Cellular RNA Is Synthesized by RNA Polymerases (2/2)

• RNA polymerase initiates and elongates the RNA product, with the chain growing in the 5’-to-3’ direction. The 3’-OH of the growing chain attacks the innermost phosphoryl (α) group of the incoming ribonucleoside triphosphate.

Now, there are three major classes of RNA, and these are important to know. The first one is messenger RNA, also called mRNA, and that's the one that we use to make proteins with. The other two kinds are called transfer RNA, which is tRNA, and ribosomal RNA, which is rRNA. And transfer RNA is essential for creating the protein strands out of the messenger RNA. Ribosomal RNA is also important for synthesizing proteins - in fact, ribosomal RNA is the main component of the ribosome, which is the complex that catalyzes the formation of protein chains. Ribosomal RNA is a good example of where RNA can be a catalytic element itself. But we'll go into that in more detail later.

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Genes Are the Transcriptional Units

• Three major classes of RNA are synthesized.

• Messenger RNA (mRNA) encodes the information to generate a protein.

• Transfer RNA (tRNA) and ribosomal RNA (rRNA) play key roles in translating mRNA information into protein.

RNA polymerase, like DNA polymerase, has a number of different subunits. And the one, major core subunit is made up of five separate components. There's two α units, a β, a β', and an ω subunit. This combines with the σ subunit which is responsible for identifying the promoter regions of these genes. And we'll talk about that in a second.

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RNA Polymerase Is Composed of Multiple Subunits

• RNA polymerase is composed of five kinds of subunits.

• The holoenzyme, consisting of α2ββ’σω subunits, initiates RNA synthesis.

• The core enzyme, composed of α2ββ’ω subunits, elongates the RNA product.

So the three stages of RNA synthesis are initiation, elongation, and termination. Basically, you start writing the RNA, you continue writing the RNA, and you stop writing the RNA. That might seem kind of trivial, but there are specific proteins that are involved in each stage of this process.

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Section 36.2 RNA Synthesis Comprises Three Stages

The three stages of RNA synthesis:

• Initiation

• Elongation • Termination

Promoter sites tell the RNA polymerase where to start polymerizing. And the promoters are specific DNA sequences that usually exist upstream of the start site. If you look at the bottom of this slide you'll see the start site is what we call base one. And then -10 (negative ten), which means 10 bases before that, is where you have one element of the promoter, and -35 (negative thirty five) bases before the start site you have another element of the promoter. Now, these are not absolute rules on this, but they are pretty common. And what we call this is a “consensus sequence”, which means that, in general, overall, this is what the sequences tend to be. But there can be a lot of other sequences that also affect the promoter area.

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Transcription Is Initiated at Promoter Sites on the DNA Template

• Promoters are specific DNA sequences that direct RNA polymerase to the proper initiation site.

• In E. coli, two DNA sequences that act as a promoter for many genes are the -10 site (Pribnow box) and the -35 sequence.

• There are variations in the sequence of the promoter for different genes. The average or consensus sequences are

• Other sequences upstream of the promoter can enhance promoter effectiveness.

Figure 36.4 Prokaryotic promoter sequences. A comparison of five sequences from prokaryotic promoters reveals a recurring sequence of TATAAT centered on position −10. The –10 consensus sequence (in red) was deduced from a large number of promoter sequences.

Here’s an example of how we might determine a consensus sequence. You have five different sequences from prokaryotic promoters represented here. At the top you can see TATGTT, centered around the negative ten base (which is not the first or the last, but one of the middle ones). And below it you have TATGGT. You can see in all of these, even though they are all different, there are some similarities. Most of them start with T. Most of them have an A in the second place. Most of them have a T in the third place. For example. So that's what we mean by a consensus sequence. The sequences are not identical, but they tend to revolve around a certain set of bases. And that's an important thing, actually - because the most effective promotion site might be TATAAT, but if you change a couple of those letters, then the promoter might be weaker. The sigma subunit might not be able to bind as well. And that can change the rate at which that gene is transcribed. And that's a very important regulatory feature. You don't want all of the genes to be transcribed at the same rate; you want some of them to be transcribed more than others, depending on what the needs are of the cell. So this is one layer where you can control the rate of transcription of these genes.

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Diagram of Prokaryotic Promoter Sequences

Figure 36.5 The RNA polymerase holoenzyme complex. Notice that the s subunit (orange) of the bacterial RNA polymerase holoenzyme makes sequence-specific contacts with the –10 and –35 promoter sequences (yellow). [From K. S. Murakami, S. Masuda, E. A. Campbell, O. Muzzin, and S. A. Darst. Science 296:1285–1290, 2002.]

Now, as we've been mentioning, the σ subunit is what recognizes these -35 and -10 elements. And in this diagram it kind of looks like you have three separate σ subunits but it's actually all one unit that is connected in the back behind this blue molecule. Now, polymerase has good affinity for DNA and that can be a problem because you don't want it to stick in the wrong place - you want it to stick where there's a promoter. So one of the things that the σ subunit does is it prevents the polymerase from sticking to the wrong place - it only sticks to where there's a promoter region. The σ subunit, I believe, is able to slide down the DNA, and that way it can scan to see if there are any promoters nearby.

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Sigma Subunits of RNA Polymerase Recognize Promoter Sites

• The sigma (σ) subunit of the holoenzyme helps the polymerase locate promoter sites.

• By decreasing the affinity of the polymerase for DNA, the σ subunit allows the enzymes to rapidly scan the DNA for a promoter.

Figure 36.6 Sigma factors act catalytically. The s factor assists the polymerase in finding the correct promoter site and then dissociates from the active enzyme to help another polymerase find the proper promoter. Thus, one s factor can aid many polymerases in locating promoters.

Once the combination of the polymerase and the σ subunit find a promoter region, then the polymerase can attach to the DNA and start the transcription of the new strand - and the σ subunit is no longer needed. So it diffuses away and is able to join with another polymerase and start the transcription of a different gene.

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Diagram Depicting Sigma Factors Acting Catalytically

• Once the promoter is located and RNA synthesis begins, the σ subunit dissociates from the enzyme to assist another polymerase in initiation.

Figure 36.7 DNA unwinding. RNA polymerase unwinds about 17 base pairs of double-helical DNA to form the open promoter complex.

The first step necessary to transcribe a gene is to unwind the DNA and expose the bases. So after a promoter is located by the polymerase, it unwinds about 16 or 17 bases to form an open complex.

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RNA Strands Grow in the 5’-to-3’ Direction

• When the promoter is initially located by the polymerase, the complex formed is called the closed promoter complex because the DNA helix is not unwound.

• RNA polymerase unwinds approximately 17 bases to form an open promoter complex in which the DNA acts as the template.

Figure 36.8 The 5¢ end of RNA. The 5¢ end of RNA has a distinct tag.

Now, RNA synthesis does not require a primer like DNA synthesis does. If you'll recall, the primer for DNA synthesis was RNA. So you're able to lay down RNA just straight without any kind of primer - which means that you start off with a triphosphate on the 5' end of the strand. And that stays that way throughout.

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Diagram of the 5’ end of RNA

Figure 36.9 A transcription bubble. A schematic representation of a transcription bubble in the elongation of an RNA transcript. Duplex DNA is unwound at the forward end of RNA polymerase and rewound at its rear end. The RNA–DNA hybrid rotates during elongation.

So once you've laid out a few of the initial bases, then you can start elongating the strand of RNA. At this point the polymerase starts moving down the DNA and unwinding a portion of it, so that it can lay down the new RNA bases, forming a RNA strand that is complementary to the DNA strand. And for a little while there is a hybrid RNA-DNA helix that is formed. But then the RNA is fed out of the RNA polymerase, and the helix is dissociated, and the DNA double helix is reformed.

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Elongation Takes Place at Transcription Bubbles That Move Along the DNA Template • Once the DNA is unwound, elongation can take place.

• The region containing the RNA polymerase, DNA, and the RNA product is called the transcription bubble.

• The transcription bubble moves along the DNA as DNA is unwound and then rewound, and the RNA product is extruded from the complex.

• A DNA–RNA hybrid helix of approximately eight nucleotides is an intermediate in RNA synthesis.

Figure 36.10 RNA–DNA hybrid separation. A model based on the crystal structure of the RNA polymerase holoenzyme shows the unwound DNA forming the transcription bubble. Notice that the RNA is peeled from the template strand and extruded from the enzyme. [Drawn from 1CDW.pdb by Adam Steinberg.]

This is a slightly more molecular detail look at the DNA and RNA and polymerase complex. And you can see, hopefully, two strands of DNA. The template strand is the red strand that would be fed through the active site. And the RNA is the green strand which is feeding out the back of this molecule. The active site there in the middle is, I believe, actually covered by other parts of the protein that are not shown in this figure.

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Diagram of RNA–DNA Hybrid Separation

And here's another representation of the same thing, showing a little bit more clearly the active site, and the nucleotide in the middle with its associated green magnesium ion. Note that the DNA has to be bent at a more or less 90 degree angle in order for it to fit into this active site.

Figure 36.11 Termination signal. A termination signal found at the 3¢ end of an mRNA transcript consists of a series of bases that form a stable stem-and-loop structure and a series of U residues.

So it'll continue synthesizing the new RNA strand until the termination stage. And one way that terminates is through the formation of a hairpin loop followed by a number of uracil residues. This hairpin loop is due to a palindromic sequence of {RNA} which matches up and complements itself. And this structure is something that interacts with the RNA polymerase, and slows it down, and causes the RNA to be detached.

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An RNA Hairpin Followed by Several Uracil Residues Terminates the Transcription of

Some Genes • Elongation continues until a termination

signal is detected. • The simplest stop signal is the transcribed

product of a segment of palindromic DNA. • The RNA complement of the DNA stop

signal forms a hairpin structure, followed by several uracil residues.

• Upon synthesis of the hairpin, the polymerase stalls, the RNA product that is weakly bound because of the rU–dA base pairs is released, and the DNA double helix reforms. This type of termination is called intrinsic termination.

DID YOU KNOW? Derived from the Greek palindromos, meaning “running back again,” a palindrome is a word, sentence, or verse that reads the same from right to left as it does from left to right; “radar” and “senile felines” are examples.

Figure 36.12 The mechanism for the termination of transcription by r protein. This protein is an ATP-dependent helicase that binds the nascent RNA strand and pulls it away from RNA polymerase and the DNA template.

Another method for terminating the RNA synthesis is the use of the rho protein (ρ protein). And that's a hexameric protein (six units) that attaches to the growing RNA strand, and climbs down it, and basically chases the RNA polymerase. And once it catches up with the RNA polymerase, it causes the transcription to stop, and the RNA to detach. This mechanism is specific to prokaryotes and is not found in eukaryotic cells.

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The Rho Protein Helps Terminate the Transcription of Some Genes

• Another type of termination signal, called protein- dependent termination, requires the protein rho (ρ).

• Rho binds to a particular sequence on the RNA product and uses the energy of ATP hydrolysis to chase down the polymerase in the transcription bubble.

• Contact with rho causes the transcription bubble to dissociate.

Figure 36.13 Primary transcript. Cleavage of this transcript produces 5S, 16S, and 23S rRNA molecules and a tRNA molecule. Spacer regions are shown in yellow.

Now, mRNA does not get modified very much after it's produced. That's necessary for the decoding of the proteins. But the other two types of RNA, rRNA and tRNA, do undergo modifications after being transcribed. You see the diagram at the bottom - that's one transcript, and it contains three rRNAs and one tRNA. So those get cut out of the larger transcript after transcription is complete. So that's one thing that’s different than mRNA. tRNA sequences need to have a CCA at the 3' end for the attachment of amino acids. And that is often not coded in the RNA, and has to be added post-transcriptionally. And finally, there are bases in both tRNA and rRNA that are modified from the original bases in several ways.

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Precursors of Transfer and Ribosomal RNA Are Cleaved and Chemically Modified After

Transcription • Although mRNA undergoes little or no modification after synthesis in bacteria,

the same is not true for rRNA or tRNA.

• Ribosomal and transfer RNA are modified as follows:

1. The final mature RNA is cleaved from a larger precursor molecule and may have other modifications.

2. Many tRNA transcripts lack the CCA sequence at the 3’ end of the strand. These nucleotides are added post-transcriptionally.

3. The bases and riboses of tRNA and rRNA are modified, for instance, by the attachment of methyl groups.

Figure 36.14 Base modifications in RNA. (A) A modified adenylate. (B) Two examples of modified uridylate.

One way of modifying bases in tRNA and rRNA is by methylation. And you'll see on the left what looks like a pretty normal adenine base, instead has got two methyl groups attached to the nitrogen on the top, in red - and that forms a different form of the base called 6-dimethyladenylate. Similarly, the base uridylate can be modified by the addition of a methyl group on the upper right, as you see, to ribothymidylate, or by changing the attachment site of the uridylate to create pseudouridylate as seen in the lower right. These modifications are important because, like proteins...proteins only have 20 amino acids and therefore use prosthetic groups to perform reactions that are not possible with those 20 amino acids. Well, these tRNAs and rRNAs are also somewhat catalytic. And, therefore, the four bases that are available for forming the RNA are not enough to perform all of the catalytic activities that these RNAs need to perform. So they need to be modified so that they can be able to conduct a larger array of activities.

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Diagram of Base Modifications in RNA

Now, let's talk about the lac operon. The lac operon is one of the canonical examples of genetic regulation. And everybody learns this one, and it's a good example, I think, of how genetic regulation occurs. So let's check it out.

You see, at the bottom, you have lactose. And that can be split into the monosaccharides galactose and glucose by the enzyme β-galactosidase. This is a very important process if you want to metabolize lactose, but if there is no lactose around then you don't really need β-galactosidase. So the lac operon is a way that the presence of lactose actually promotes the transcription and translation of the β- galactosidase gene, therefore increasing its number and activity and ability to digest lactose. It simultaneously increases the production of two other proteins, a permease and a transacetylase. So the combination of all of these genes and the control factors is what is called an operon. So this would be the lac operon.

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Section 36.3 The lac Operon Illustrates the Control of Bacterial Gene Expression

Learning objective 2: Describe how transcription is controlled in bacteria. • Transcription is a regulated process. For instance, in E. coli,

the gene for β-galactosidase, which metabolizes lactose, is minimally transcribed unless lactose is present.

• In the presence of lactose, the genes for β-galactosidase as well as two other enzymes—galactoside permease and thiogalactoside transacetylase―are expressed.

• Such a coordinated unit is called an operon, and in the case of lactose metabolizing enzymes, the unit is called the lac operon.

Figure 36.16 b-Galactosidase induction. The addition of lactose to an E. coli culture causes the production of b-galactosidase to increase from very low amounts to much larger amounts. The increase in the amount of enzyme parallels the increase in the number of cells in the growing culture. b-Galactosidase constitutes 6.6% of the total protein synthesized in the presence of lactose.

This is an overly simplified diagram, but it shows that the β-galactosidase level is very low before you add lactose to the media. And then it starts being produced, and it gets produced until lactose is removed from the media.

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Graph of β-galactosidase Induction

Figure 36.17 Operons. (A) The general structure of an operon as conceived by Jacob and Monod. (B) The structure of the lactose operon. In addition to the promoter, p, in the operon, a second promoter is present in front of the regulator gene, i, to drive the synthesis of the regulator.

So let's take a look at the lac operon at the DNA level. We have a regulator gene in the front, and then some control sites, and then the structural genes that are transcribed later. And if you look at the bottom, the specific lac operon, we have a promoter unit (that's what the p stands for) on the left. And then the i is an inhibitor unit {i = inhibitor}. And then there's a second promoter unit, which is the one that transcribes the other three genes. But right after that promoter unit, is an o, which is the operator unit {o = operator}. And that is where the inhibitor will bind and prevent the transcription of these genes.

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An Operon Consists of Regulatory Elements and Protein-Encoding Genes

• The DNA components of the regulatory system for an operon consist of a regulator gene; an operator site, which includes a promoter; and structural genes.

• In the lac operon, the regulatory gene encodes a protein called the lac repressor that binds to the operator site in the absence of lactose and prevents transcription of the structural genes.

Now, it's important to realize this is not an on-off switch, so to speak. There is a level of different activities that this operon can participate in. And even when there is no lactose present, it still produces a very small amount of the β-galactosidase. That β- galactosidase is floating around and present in case some lactose happens to arrive. Then the β-galactosidase can process it. And normally what it does is break it into galactose and glucose, but there is also a side reaction which produces a molecule called allolactose. Allolactose is the signal that tells the lac operon to start producing.

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Ligand Binding Can Induce Structural Changes in Regulatory Proteins

• When lactose is present, the small amount of β-galactosidase in the cell converts it to galactose and glucose.

• However, the enzyme generates allolactose in a side reaction.

• Allolactose is the inducer of the lac operon. Upon binding allolactose, the lac repressor undergoes a structural change that greatly reduces its affinity for DNA.

• RNA polymerase, which binds at the promoter, can then transcribe the structural genes of the operon.

Figure 36.18 The induction of the lac operon. (A) In the absence of lactose, the lac repressor binds DNA and represses transcription from the lac operon. (B) Allolactose or another inducer binds to the lac repressor, leading to its dissociation from DNA and to the production of lac mRNA.

So here's a diagram of the lac operon up at the top. And you can see the “i” molecule is being transcribed all the time. That “i” molecule is a repressor, and that binds to the operator site of the lac operon. So there's always these “i” molecules floating around. That's the lac repressor. And an actual image of what it looks like, more realistically, on the bottom, shows you that it actually creates a loop in the DNA and that prevents the polymerase from sliding down and transcribing the remaining genes. So even if the polymerase assembles at the p, at the promoter unit of this lac operon, it can't proceed.

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Diagram of the Induction of the lac Operon (1/2)

More realistic:

A molecule of allolactose, however, functions as an inducer. And you can see in the center/near the center of this diagram the red word "inducer." That's your allolactose. And what that does is it attaches to the inhibitor molecule (or molecules actually) which is the lac repressor. And when it attaches then it induces a conformational change, and the lac repressor no longer sticks to the DNA. With the lac repressor gone, the polymerase is able to continue down and transcribe the remaining genes in the operon. Those genes are β-galactosidase, permease, and transacetylase.

I've got some pictures of those at the bottom. Β-galactosidase is the one that we think is the most important. The permease actually is a protein that goes into the membrane of the cell and allows more lactose in, because if there is lactose around, then "let's get it, and eat it." Otherwise there's no real use for this lactose permease and so its transcription is kept to a minimum - not off, but to a minimum. You know, it allows a little bit of lactose in, but once that lactose is detected by this circuit, then it will start letting in a lot more. The third gene codes for a transacetylase, or acetyltransferase. And we still don't know what that does, as far as why it's part of the lac operon. That just goes to show you that we're still learning these things. The lac operon's been studied for fifty years and we still don't know what this one other protein does. So this is why biology and biochemistry is still exciting and interesting and needs to have people studying it. Similarly you can see in the lower right, the structure there is actually not taken off of real data - it's an imagined structure

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Diagram of the Induction of the lac Operon (2/2)

Role unknown

Structure unsolved

because we don't have the structure yet. So there is work to be done even on these very basic things.

Figure 36.19 The binding site for catabolite activator protein (CAP). This protein binds as a dimer to an inverted repeat that is at the position −61 relative to the start site of transcription. The CAP-binding site on DNA is adjacent to the position at which RNA polymerase binds.

So those were the basic elements of the lac operon - but we want to discuss here another thing which can happen which is the promotion of transcription. Now, of course, if the lac operon is inhibited then there won't be any transcription. But if it's not inhibited, then it can be transcribed at varying rates. And one way that you can increase the rate of transcription is with the activator protein CAP, which stands for catabolite activator protein. And that's a dimer, just like the lac repressor was. And that can attach to the DNA, and that helps to recruit the RNA polymerase.

This occurs when there is a low level of glucose around. And that causes the concentration of cyclic AMP to increase in the cell. The cyclic AMP, or cAMP, binds to this CAP protein, and that causes it to bind to the DNA, and that stimulates the activity of the polymerase. So it kind of makes sense – “if there's not much glucose around, let's ramp up our activity and see if we can find some lactose.” Because, remember, the lac operon also produces a permease which can let more lactose in.

But when the glucose concentration increases, then that cyclic AMP concentration falls, and the CAP dissociates from the DNA, and therefore it's like, "Well, okay,

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Transcription Can Be Stimulated by Proteins That Contact RNA Polymerase

• Expression of the lac operon can also be stimulated by proteins such as the catabolite activator protein (CAP).

• In E. coli, the concentration of cAMP increases when the concentration of glucose decreases.

• Cyclic AMP binds the CAP, and the cAMP–CAP complex binds to DNA near the start site of the lac operon and stimulates the activity of RNA polymerase.

• When glucose concentration increases, the cAMP concentration falls and the lac operon is not expressed, a process called catabolite repression.

there's a lot of glucose around, we don't really need the lactose as much, so let's cool it down a little bit."

Note on the right that CAP binding induces a bend in the DNA. I'm not sure if that has any functional significance, but I just thought it was interesting because normally you see all these DNA molecules and they're all straight and these things kind of sit on them. Well, in the real molecular world, things are very flexible and are moving all the time. These CAP proteins sit on the DNA about 60 base pairs before the start site of transcription, so right adjacent to where the promoter sequence is, and that's where they can help recruit polymerase.

And you guys know how that works, right? I mean, you've got your DNA which is already recognized by your σ subunit - so what did this CAP do? Well, it just provides more surface area that is specifically interacting with the RNA polymerase. And so if RNA polymerase comes by, it is more likely to stick at that spot because it will interact with the CAP as well as the σ subunit, and that gives you twice as much chance for the polymerase to actually bind.

Operons can be used to facilitate a certain activity called “quorum sensing,” which means the bacteria, and this is like a bacterial thing, bacteria cells only induce the production of certain genes when there are a whole bunch of them together. And the example we're going to use for this is Vibrio fischeri which is a bacteria that lives inside the bobtail squid. And that's where the process of quorum sensing was discovered. So these bacteria produce a protein called luciferase which has bioluminescence. So it glows in the dark. Now, an individual bacterium would not be visible if it produced this luciferase. So there’s really no point in making it unless there is a whole bunch of these bacteria together. Now, for the squid - it's actually helpful for them to be a little bit lit up, because predators would be coming from below, and against the light in the sky, the body of the squid would look dark, and would be easy to spot. But with bioluminescence, the squid's body becomes less easy to spot. So it's a form of camouflage. So the bacterial cells, the squid likes them, and actually has an organ inside which is specifically suited to the growth of these bacterial cells. So these bacteria float around in the ocean and then get concentrated inside the light organ of the squid. Now, they're always producing this signaling molecule, and this signaling molecule is something that, like the lactose in the lac operon, will trigger an operon inside the bacteria to produce luciferase. But if they're sitting alone, then these molecules are diffusing away all of the time, and the concentration is not very high. But if you get a whole bunch of these bacteria together in one place, and they are all together secreting this signaling molecule, then the concentration rises to a point where it's high enough that it can induce the operon. A similar circumstance

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Clinical and Biological Insight: Many Bacterial Cells Release Chemical Signals that Regulate Gene

Expression in Other Cells CLINICAL AND BIOLOGICAL INSIGHT Many Bacterial Cells Release Chemical Signals that Regulate Gene Expression in Other Cells

• Quorum sensing is a type of interaction among bacterial cells originally discovered in Vibrio fischeri, which live inside a light organ of bobtail squid.

• In a symbiotic relationship, the bacteria synthesize luciferase, an enzyme that generates bioluminesce that protects the squid from predators, in return for a protected environment.

• The bacterial cells release an autoinducer into the environment that, upon uptake by neighboring cells, stimulates the expression of the luciferase gene. Since each cell produces only a small amount of autoinducer, the amount of autoinducer present enables cells to sense the population density—hence, the name quorum sensing.

• Quorum sensing allows bacteria to form biofilms, large communities of bacteria that are resistant to the host immune response as well as to many antibiotics.

occurs with the formation of biofilms, which is when a whole bunch of bacteria get together, they actually can sense each other's presence, and start producing different sorts of materials in order to protect themselves as a colony. For example the cells on the outside of the colony might produce mucus to prevent enemies of the colony from getting in.

Figure 36.20 Bobtail squid. The bobtail squid, only 1 to 8 cm in length, inhabits the coastal waters of the Pacific and provides safe haven for the bacterium Vibrio fischeri. [David Fleetham/Alamy.]

This is just a picture of that bobtail squid. It's pretty gorgeous. That’s why I thought I would share it with you.

Created by Brett Barbaro

Image of a Bobtail Squid

And now this is very interesting. Riboswitches. So some bacterial mRNA have sequences in them that can directly detect other molecules. So we see here, on the left, we've got a diagram of mRNA which has a couple of hairpin loops in it. And one of those hairpin loops contains the AUG which is the start signal for translation - that's highlighted in yellow - as well as this ribosomal binding site, which is highlighted in pink. Now, when these two sequences are in the hairpin loop, they are not accessible to ribosomes, and therefore this gene cannot be translated. But if a small molecule comes that interacts with this mRNA, then it can change the structure of this loop, and expose the ribosomal binding site and the start site for translation. So in that way, the small molecule actually basically “turns on” the mRNA. So this is an interesting example of how mRNA, and RNA in general, can have some very interesting catalytic and functional aspects to it.

Created by Brett Barbaro

Some Messenger RNAs Directly Sense Metabolite Concentrations

• Some bacterial mRNA can sense environmental signals as well as encode proteins.

• These mRNA have special structures, called riboswitches, that bind small molecules that cause a structural change in the riboswitch and terminate the synthesis of the mRNA.

http://2011.igem.org/wiki/images/e/eb/Wits_Overview_Riboswitch_Considerations_2.jpg

Example of translation triggering: