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recognition site

The detailed mechanism by which repressors prevent transcription varies considerably and is often unknown. The repressor sometimes blocks the binding of RNA polymerase to the promoter, simply by getting in the way (steric hindrance). An example is the well-studied CI repressor of bacteriophage lambda. Sometimes the repressor may bind further downstream, inside the structural gene. In this case, RNA polymerase can still bind to the promoter but is prevented from moving forward and transcribing the gene. Sometimes, even though their binding sites in the DNA sequence overlap, the RNA polymerase and the repressor both bind the DNA simultaneously. [Remember that the DNA double helix is 3-dimensional and that two proteins may therefore bind to the same linear segment, if they occupy separate locations around its surface.] Indeed, an example of this is the LacI repressor. In this case the RNA polymerase actually binds more tightly in the presence of repressor, but is locked in place and cannot open the DNA to initiate transcription.

Small molecules may control gene expression by binding to regulatory proteins.

Many Regulator Proteins Bind Small Molecules and Change Shape

Whether a regulator protein is an activator or a repressor, it needs a signal of some sort. One of the most common ways to do this is by using some small molecule that fits into a binding site on the regulatory protein (Fig. 6.14). This is called the signal molecule. In the case of using a nutrient for growth, an obvious and common choice is the nutrient molecule itself. [In prokaryotes the DNA binding protein often binds the signal molecule directly. In eukaryotes, where the DNA is inside the nucleus, things are often more complex, and multiple proteins are involved. The signal molecule is often bound by proteins in the cell membrane or cytoplasm and the signal is then transmitted to the nucleus. The DNA binding protein itself normally stays in the nucleus and upon receiving the signal, is converted to its DNA-binding form by phosphorylation.]

When a regulator protein binds its signal molecule, it changes shape (Fig. 6.14). Regulator proteins have two alternative forms, the DNA-binding form and the non-binding form. Binding or loss of the signal molecule causes the larger protein to flipflop between its two alternative shapes. Proteins that change in activity by changing signal molecule Small molecule that exerts a regulatory effect by binding to a regulatory protein

Transcription in Eukaryotes Is More Complex 145

FIGURE 6.15 Regulator Binds at an Inverted Repeat—Principle

At sites where regulator proteins bind there is often an inverted repeat with both DNA strands participating as shown. If the subunits of regulator protein are identical, they each recognize one of the inverted repeats and pair so that the same regions of each subunit face each other.

FIGURE 6.15 Regulator Binds at an Inverted Repeat—Principle

At sites where regulator proteins bind there is often an inverted repeat with both DNA strands participating as shown. If the subunits of regulator protein are identical, they each recognize one of the inverted repeats and pair so that the same regions of each subunit face each other.

Recognition sites on DNA are often inverted repeats. Separate subunits of the regulator protein each bind one of the repeat sequences.

shape in this manner are called allosteric proteins. Examples include some enzymes, transport proteins and regulators. Allosteric proteins have multiple subunits that change shape in concert (Fig. 6.14). Usually there is an even number of subunits, most often two or four. All of the subunits bind the signal molecule and then they all change shape together.

Since there is an even number of protein subunits, the recognition site on the DNA for regulator proteins is often duplicated. In this case, the recognition site is usually an inverted repeat, often referred to as a palindrome. This is because the subunits of the regulator protein bind to each other head to head rather than head to tail (Fig. 6.15). Consequently, the two protein molecules are pointing in opposite directions. Because they have identical binding sites for DNA, they recognize the same sequence of bases but in opposite directions on the two strands of the DNA. The two half-sites are usually separated by a spacer region of several bases, whose identity is free to vary. The two half-sequences of such recognition sites are not always exact matches.

One regulator protein subunit binds to the recognition sequence on the template strand of the DNA double helix, and its partner binds to the same sequence but on the non-template strand of the DNA pointing in the opposite direction. This is simpler in practice than it sounds, precisely because the DNA molecule is helical. Although the two recognition sequences are on different strands of DNA, they end up on the same face of the DNA molecule due to its helical twisting (Fig. 6.15).

An example of a palindromic recognition site is the lac operator sequence which is bound by the LacI repressor (a tetramer). This sequence runs from -6 to +28 relative to the start of transcription. It is not exactly symmetrical. The two half sequences are: TGTGTGgAATTGTgA and, running in the opposite sense on the other strand, TGTGTGaAATTGTtA (capital letters indicate matching bases). The two half-sites are separated by five base pairs. The left hand half of this site binds the LacI protein more strongly than the right hand side. A stronger operator sequence could be generated by changing the right hand half-site to exactly match the left.

Eukaryotes have three RNA polymerases that specialize in which type of genes they transcribe.

Transcription in Eukaryotes Is More Complex

Since typical eukaryotic cells have 10 times as many genes as do bacteria, the whole process of transcription and its regulation is more complex. For a start, eukaryotes have three different RNA polymerases, unlike bacteria which have just one. The three RNA polymerases transcribe different categories of nuclear genes. In addition, mitochondria and chloroplasts have their own RNA polymerases, which resemble the bacterial enzyme.

allosteric protein Protein that changes shape when it binds a small molecule

Non-transcribed space

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