Transcription in Prokaryotes

Transcription in Prokaryotes

RNA Polymerase in Escherichia coli

The enzyme that synthesizes RNA is called RNA polymerase, and the most extensively studied one was isolated from E. coli. The molecular weight of this enzyme is about 470,000 Da, and it has a multisubunit structure. Five different types of subunits, designated α, ω, β, β', and σ, have been identified. The actual composition of the enzyme is α₂ωββ'σ . The σ-subunit is rather loosely bound to the rest of the enzyme (the α₂ωββ' portion), which is called the core enzyme. The holoenzyme consists of all the subunits, including the σ-subunit.

What do the subunits of RNA polymerase do?

The σ-subunit is involved in the recognition of specific promoters, whereas the β-, β'-, α-, and ω-subunits combine to make the active site for polymerization.The mechanism of the polymerization reaction is an active area of research.

Which of the DNA strands is used in transcription?

Figure 11.1 shows the basics of information transfer from DNA to protein. Of the two strands of DNA, one of them is the template for RNA synthesis of a particular RNA product. RNA polymerase reads it from 3' to 5'. This strand has several names. The most common is the template strand, because it is the strand that directs the synthesis of the RNA. It is also called the antisense strand, because its code is the complement of the RNA that is produced. It is sometimes called the (–) strand by convention. The other strand is called the coding strand because its sequence of DNA will be the same as the RNA sequence that is produced (with the exception of U replacing T). It is also called the sensestrand, because the RNA sequence is the sequence that we use to determinewhat amino acids are produced in the case of mRNA. It is also called the (+)strand by convention, or even the nontemplate strand. For our purposes, wewill use the terms template strand and coding strand throughout. Because the DNA in the coding strand has the same sequence as the RNA that is produced, it is used when discussing the sequence of genes for proteins or for promoters and controlling elements on the DNA.

The core enzyme of RNA polymerase is catalytically active but lacks specific-ity. The core enzyme alone would transcribe both strands of DNA when only one strand contains the information in the gene. The holoenzyme of RNA polymerase binds to specific DNA sequences and transcribes only the correct strand. The essential role of the σ-subunit is recognition of the promoter locus (a DNA sequence that signals the start of RNA transcription;). The loosely bound σ-subunit is released after transcription begins and about 10 nucleotides have been added to the RNA chain. Prokaryotes can have more than one type of σ-subunit. 

The nature of the σ-subunit can direct RNA poly-merases to different promoters and cause the transcription of various genes to reflect different metabolic conditions.

Promoter Structure

Even the simplest organisms contain a great deal of DNA that is not transcribed. RNA polymerase must have a way of knowing which of the two strands is the template strand, which part of the strand is to be transcribed, and where the first nucleotide of the gene to be transcribed is located.

How does RNA polymerase know where to begin transcription?

Promoters are DNA sequences that provide direction for RNA polymerase. The promoter region to which RNA polymerase binds is closer to the 3' end of the template strand than is the actual gene for the RNA to be synthesized. The RNA is formed from the 5' end to the 3' end, so the polymerase moves along the template strand from the 3' end to the 5' end. However, by convention, all control sequences are given for the coding strand, which is 5' to 3'. The binding site for the polymerase is said to lie upstream of the start of transcription, which is farther to the 5' side of the coding strand. This is often a source of confusion to students, and it is important to remember the correct orientation. The promoter sequence will be given based on the coding strand, even though the RNA polymerase is actually binding to the template strand. Promoters are upstream, which means to the 5' side of the coding strand and to the 3' side of the template strand.

Most bacterial promoters have at least three components. Figure 11.2 shows some typical promoter sequences for E. coli genes. The component closest to the first nucleotide to be incorporated is about 10 bases upstream. Also by con-vention, the first base to be incorporated into the RNA chain is said to be at position +1 and is called the transcription start site (TSS). All the nucleotides upstream from this start site are given negative numbers.

Because the first promoter element is about 10 bases upstream, it is called the –10 region, but is also called the Pribnow box after its discoverer. After the Pribnow box, there are 16 to 18 bases that are completely variable. The next promoter element is about 35 bases upstream of the TSS and is simply called the –35 region or –35 element. 

An element is a general term for a DNA sequence that is somehow important in controlling transcription. The area from the –35 element to the TSS is called the core promoter. Upstream of the core promoter can be an UP element, which enhances the binding of RNA polymerase. UP elements usually extend from –40 to –60. The region from the end of the UP element to the transcription start site is known as the extendedpromoter.

The base sequence of promoter regions has been determined for a number of prokaryotic genes, and a striking feature is that they contain many bases in common. These are called consensus sequences. Promoter regions are A–T rich, with two hydrogen bonds per base pair; consequently, they are more easily unwound than G–C-rich regions, which have three hydrogen bonds per base pair. Figure 11.2 shows the consensus sequences for the –10 and –35 regions.

Even though the –10 and –35 regions of many genes are similar, there are also some significant variations that are important to the metabolism of the organism. Besides directing the RNA polymerase to the correct gene, the pro-moter base sequence controls the frequency with which the gene is transcribed. Some promoters are strong, and others are weak. A strong promoter binds RNA polymerase tightly, and the gene therefore is transcribed more often. In general, as a promoter sequence varies from the consensus sequence, the bind-ing of RNA polymerase becomes weaker.

Chain Initiation

The process of transcription (and translation as well, as we will see is usually broken down into phases for easier studying. The first phase of transcription is called chain initiation, and it is the part of transcription that has been studied the most. It is also the part that is the most controlled. 

Chain initiation begins when RNA polymerase (RNA pol) binds to the promoter and forms what is called the closed complex (Figure 11.3). The σ-subunit directs the polymerase to the promoter. It bridges the –10 and –35 regions of the promoter to the RNA polymerase core via a flexible “flap” in the σ-subunit. 

Core enzymes lacking the σ-subunit bind to areas of DNA that lackpromoters. The holoenzyme may bind to “promoterless” DNA, but it dissoci-ates without transcribing.

Chain initiation requires formation of the open complex. Recent studies show that a portion of the β' and the σ-subunits initiate strand separation, melting about 14 base pairs surrounding the transcription start site. A purine ribonucleoside triphosphate is the first base in RNA, and it binds to its comple-mentary DNA base at position +1. Of the purines, A tends to occur more often than G. This first residue retains its 5'-triphosphate group (indicated by ppp in Figure 11.3).

Chain Elongation

After the strands have separated, a transcription bubble of about 17 base pairs moves down the DNA sequence to be transcribed (Figure 11.3), and RNA polymerase catalyzes the formation of the phosphodiester bonds between the incorporated ribonucleotides. When about 10 nucleotides have been incorporated, the σ-subunit dissociates and is later recycled to bind to another RNA polymerase core enzyme.

The transcription process supercoils DNA, with negative supercoiling upstream of the transcription bubble and positive supercoiling downstream, as shown in Figure 11.4. Topoisomerases relax the supercoils in front of and behind the advancing transcription bubble. The rate of chain elongation is not constant. The RNA polymerase moves quickly through some DNA regions and slowly through others. It may pause for as long as one minute before continuing.

Instead of finishing every RNA chain, RNA polymerase actually releases most chains near the beginning of the process, after about 5 to 10 nucleotides have been assembled, in a process called abortive transcription. The cause of abortive transcription is the failure of RNA polymerase to break its own bonds to the promoter via the σ-subunit. Studying this process has led to the current model of the mechanism of transcription. In order for chain elongation to occur, the RNA polymerase must be able to launch itself off the promoter. Given the tight binding between the σ-subunit and the promoter, this requires substantial energy. Figure 11.5 shows the current model of the open complex that allows progression into chain elongation. The RNA polymerase is bound tightly to the DNA promoter. It “scrunches” the DNA into itself, causing torsional strain of the separated DNA strands. Like a bow loading up with potential energy as the bowstring is pulled, this provides the energy to allow the polymerase to break free. 

Chain Termination

Termination of RNA transcription also involves specific sequences downstream of the actual gene for the RNA to be transcribed. There are two types of termination mechanisms. The first is called intrinsic termination, and it is controlled by specific sequences called termination sites. The termination sites are characterized by two inverted repeats spaced by a few other bases (Figure 11.6). Inverted repeats are sequences of bases that are complementary, such that they can loop back on themselves. The DNA then encodes a series of uracils. When the RNA is created, the inverted repeats form a hairpin loop. This tends to stall the advancement of RNA polymerase. At the same time, the presence of the uracils causes a series of A–U base pairs between the template strand and the RNA. A–U pairs are weakly hydrogen-bonded compared with G–C pairs, and the RNA dissociates from the transcription bubble, ending transcription.

The other type of termination involves a special protein called rho (ρ). Rho-dependent termination sequences also cause a hairpin loop to form. In this case, the ρ protein binds to the RNA and chases the polymerase, as shown in Figure 11.7. When the polymerase transcribes the RNA that forms a hairpin loop (not shown in the figure), it stalls, giving the ρ protein a chance to catch up. When the ρ protein reaches the termination site, it facilitates the dissociation of the transcription machinery. The movement of the ρ protein and the dissociation require ATP.

Summary

Prokaryotic transcription is catalyzed by RNA polymerase, which is a 470,000-Da enzyme with five types of subunits: α, ω, β, β', and σ.

RNA polymerase moves along the template strand of DNA and produces a complementary RNA sequence that matches the coding strand of DNA.

RNA polymerase recognizes specific DNA sequences called promoters and binds to the DNA. The promoters tell the polymerase which DNA should be transcribed.

The σ-subunit is loosely bound to RNA polymerase and is involved in promoter recognition.

Transcription can be divided into three parts-initiation, elongation, and termination.