Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin

exception of some archaea (see Bukau and Horwich, Suggested Reading). These chaperones are highly conserved evolutionarily. Chaperones in this family are called the Hsp70 proteins because they are about 70 kDa in size and because more of them are made (along with many other proteins) if cells are subjected to a sudden increase in temperature, or “heat shock” (see chapter 12); other stresses that denature proteins (such as ethanol) can have the same effect. Synthesis of chaperones increases after such stresses to help refold proteins that have been denatured by the environmental stress, although they also help to fold proteins under normal conditions. The Hsp70 type of chaperone was first discovered in E. coli, where it was given the name DnaK because it is required to assemble the DNA replication apparatus of phage λ and so is required for λ DNA replication. This name for the Hsp70 chaperone in E. coli is still widely used in spite of being a misnomer, because the chaperone has nothing directly to do with DNA but functions more generally in protein folding. In its role as a heat shock protein, the DnaK protein of E. coli also functions as a cellular thermometer, regulating the synthesis of other proteins in response to heat shock (see chapter 12).

To understand how Hsp70 chaperones, including DnaK, help fold proteins, it is necessary to understand something about the structure of most proteins. Proteins are made up of chains of amino acids that are folded up into well-defined structures, which are often rounded or globular. The amino acids that make up proteins can be charged, polar, or hydrophobic (see the inside front cover for a list). Amino acids that are charged (either acidic or basic) or polar tend to be more soluble in water and are called hydrophilic (water loving). Amino acids that are not charged or polar are hydrophobic (water fearing) and tend to be in the inside of the globular protein among other hydrophobic amino acids and away from the water on the surface. If the hydrophobic amino acids are exposed, they tend to associate with hydrophobic amino acids on other proteins and cause the proteins to precipitate. This is essentially what happens when you cook an egg. High temperatures cause the proteins in the egg to unfold, exposing their hydrophobic regions, which then associate with each other, causing the proteins to precipitate into a solid white mass.

Figure 2.36 Polarity in transcription of a polycistronic mRNA transcribed from pYZ. (A) The rut site in gene Y is normally masked by ribosomes translating the gene Y mRNA. (B) If translation is blocked in gene Y by a mutation that changes the codon CAG to UAG (boxed in red), the ρ factor can bind to the mRNA and cause transcription termination before the RNA polymerase reaches gene Z. (C) Only fragments of the gene Y protein and mRNA are produced, and gene Z is not even transcribed into mRNA.

      The Hsp70-type chaperones help proteins fold by binding to the hydrophobic regions in denatured proteins and nascent proteins as they emerge from the ribosome and keeping these regions from binding to each other prematurely as the protein folds. The Hsp70 proteins have an ATPase activity that, by cleaving bound ATP to ADP, helps the chaperone to sequentially bind to, and dissociate from, the hydrophobic regions of the protein they are helping to fold. The Hsp70-type chaperones are directed in their protein-folding role by smaller proteins called cochaperones. The major cochaperones in E. coli were named DnaJ and GrpE, again for historical reasons. The DnaJ cochaperone helps DnaK to recognize some proteins and to cycle on and off of the proteins by regulating its ATPase activity. It can also sometimes function as a chaperone by itself. The GrpE protein is a nucleotide exchange protein that helps regenerate the ATP-bound form of DnaK from the ADP-bound form, allowing the cycle to continue.

      TRIGGER FACTOR AND OTHER CHAPERONES

      Given the prevalence and central role of DnaK in the cell, it came as a surprise that E. coli mutants that lack DnaK still multiply, albeit slowly. In fact, the only reason they are sick at all is because they are making too many copies of the other heat shock proteins, since DnaK also regulates the heat shock response (see chapter 12). One reason why cells lacking DnaK are not dead is that other chaperones can substitute for it. One of these is trigger factor. This type of chaperone has so far been found only in bacteria, and much less is known about it. It binds close to the exit pore of the ribosome and helps proteins fold as they emerge from the ribosome. It is also a prolyl isomerase. Of all the amino acids, only proline has an asymmetric carbon, which allows it to exist in two isomers. Trigger factor can convert the prolines in a protein from one isomer to the other. There are many other examples of chaperones that act as prolyl isomerases.

Another set of chaperones, including ClpA, ClpB, and ClpX, form cylinders and unfold misfolded proteins by sucking them through the cylinder. This takes energy, which is derived from cleavage of ATP. Some of them, including ClpA and ClpX, can also feed the unfolded proteins directly into an associated protease called ClpP, which degrades the unfolded protein. Association with ClpP switches the function of ClpA and ClpX from protein folding to protein degradation. ClpB, another cylindrical chaperone, does not associate with a protease but seems to cooperate with the small heat shock proteins IbpA and IbpB to help redissolve precipitated proteins so that they can be refolded by DnaK (see Mogk et al., Suggested Reading).

      CHAPERONINS

In addition to the relatively simple protein chaperones, cells contain much larger structures that help proteins fold. These large structures are called chaperonins, and they exist in all forms of life, including the archaea and eukaryotes. They are composed of two large cylinders with hollow chambers held together back to back with openings at their ends (Figure 2.37). They help fold a misfolded protein by taking it up into one of the chambers. A cap, called a cochaperonin, is then put on the chamber, and the protein folds within the more hospitable environment of the chamber. A more detailed model for what happens in the chamber and how this helps a protein fold is suggested by the structure (see Wang and Boisvert, Suggested Reading). When the misfolded protein is first taken up, the lining of the chamber consists of mostly hydrophobic amino acids that bind to the exposed hydrophobic regions of the misfolded protein. When the cochaperonin cap is put onto this chamber (the cis-chamber) and the bound ATP is cleaved to ADP, the lining may switch to being mostly hydrophilic amino acids, driving the more hydrophobic regions of the misfolded protein to the interior of the protein, where they usually reside in the folded protein. Binding of ATP and an unfolded protein to the other chamber (the trans-chamber) causes the cap to come off the cis-chamber, releasing the folded protein and preparing the other chamber to take up the misfolded protein and take its turn being the cis-chamber. This process takes a lot of energy, and a number of ATP molecules (one for each subunit of one chamber of the chaperonin, which is seven in E. coli [see below]) are cleaved to ADP in each cycle. It is a mystery why chaperonins have two chambers and why the folding has to alternate between the two chambers. Chaperonins composed of only one chamber function more poorly, although they might retain some of their activity (see Sun et al., Suggested Reading). The communication to one chamber that ATP and unfolded protein are bound to the opposite chamber is one of the most remarkable examples of allosterism known, especially considering that the chambers, while touching back to back, are composed of separate polypeptides.

The first chaperonin was discovered in E. coli, where it was named GroEL because it helps