Gene Regulation at Transcriptional Termination

In the previous article we saw how RNA polymerase determines how to start transcription. In this article, we will examine how it decides to end transcription.

Before we dive in, however, let’s take some time to remind ourselves of the lay of the land. So far, we are well familiar with the happenings at the promotor. As you should recall, that is where RNA polymerase binds. Further upstream at the distal elements is where you might find binding proteins like pTrpR. The arrow immediately to the right of the proximal promotor is the transcriptional start site we refer to as “+1”. Further downstream where you see the second arrow is where you will find the start codon (AUG). RNA polymerase begins to transcribe at that +1 nucleotide and continues until it gets to a transcriptional termination signal (the second arrow in the green coding region). Two different mechanisms are responsible for almost all termination of transcription in prokaryotes. They are intrinsic (or rho independent termination) and extrinsic (or rho dependent termination).

Intrinsic Termination

As the RNA is elongated during transcription, it has the ability to base-pair with itself to form secondary structures called “hairpin loops”. A hairpin loop can even be formed within the RNA polymerase complex due to GC-rich inverted repeat regions. Guanine-cytosine (GC) bonds are strong and stable since they have 3 hydrogen bonds compared to adenine-thymine (AT) interactions with only two hydrogen bonds.

A hairpin loop under the RNA polymerase complex can dramatically slow down the rate at which an RNA polymerase transcribes but is generally not enough to cause it to stop/terminate transcribing. However, if the hairpin is followed by a series of uracil (usually 6 or more), RNA polymerase will fall off the DNA template and transcription terminates. The separation is associated with the weak interaction that uracil forms with complementary adenines in the DNA template strand.

Attenuation – The trp Operon

In the previous lesson you learned that the Trp operon is controlled by an operator that can be turned off or on depending on whether or not pTrpR is binding to the DNA. However, there is more to the story. Consider the diagram below showing the Trp operon with an open stretch of DNA. After the operator (O) there is a region that is labelled as 1, 2, 3, and 4. These regions have the capability of base pairing with each other.

  1. Region 1 can base pair with region 2
  2. Region 2 can base pair with region 3
  3. Region 3 can base pair with region 4

Region 4 is followed by a run of U’s in an RNA transcript. Whenever regions 3 and 4 bind, transcription is terminated (an intrinsic terminator is formed as you should recall).

The same regions are illustrated in the following diagram in more detail. Notice the run of uracils after region 4. You should also notice that region 1 contains the tryptophan codon at two places.

Trp operon Attenuation Regions

Why is this significant? For that, you need to look at the next diagram.

The diagram shows transcription and translation of the TrpL gene, both taking place at the same time. This allows for feedback from ribosomes directly to RNA polymerases in prokaryotes in a way that cannot be accomplished in eukaryotes.

As you can see, in this diagram, tryptophan levels are low. When this happens, the ribosomes are not able to find tryptophan to add to the growing polypeptide chain. Hence it will pause. During this pause, region 1 of the RNA gets covered by the stalled ribosome. Since region 1 is covered, it cannot base pair with region 2. Hence regions 2 and 3 will base pair instead. This base pairing will cause the RNA polymerase to slow down but will not kick off the RNA polymerase. Instead, the RNA polymerase will eventually continue downstream to express the genes needed for tryptophan production.

Now let’s see what happens when tryptophan levels are high. For that, check out the next diagram.

Notice that with abundant tryptophan the ribosomes will bring in the needed tryptophan and incorporate them into the leader peptide (pTrpL). After that the ribosome arrives at a translational stop codon where it pauses and, in the process, covers regions 1 and 2. In the meantime, regions 3 and 4 forms a hairpin loop. Remember that immediately following region 4 is a string of uracils that together with the hairpin loop creates an intrinsic termination signal. Therefore, when tryptophan levels are high, an intrinsic termination event prevents RNA polymerase from transcribing the genes required to make tryptophan.

Extrinsic Termination

Extrinsic termination utilizes a protein called rho. Rho is a hexamer which binds to specific RNA sequences known as rho-binding sites. Once rho has bound to an RNA it burns ATP to move 5′ to 3′ at a rate of 200 nucleotides per second (at least twice as fast as RNA polymerase) unless blocked by translating ribosomes. When rho does catch up to and bump into a transcribing RNA polymerase, RNA polymerase gets the signal to stop transcription.

The application of the rho factor can be observed in the lac operon. If the lacZ is defective, it causes the ribosome to stall in translation allowing the RNA polymerase to pull well ahead of the ribosome. This widens the gap between them and leaves a “naked” region on the DNA upon which rho can bind. Once rho binds to the RNA, it races towards the RNA polymerase, bumps into it, and causes it to fall off the DNA, terminating transcription. This is a brilliant move since any defect in the DNA that prevents the formation of one of the proteins would make it of no use to make the others. All must be present together.

Reference: Krane, D. 2021. Bio 2110 Molecular Biology Video Lecture. Wright State University – Lake Campus.

Author

  • Courtney Simons

    Dr. Courtney Simons has served as a food science researcher and educator for over a decade. He holds a Bachelor of Science in Food Science and a Ph.D. in Cereal Science from North Dakota State University.