INTRODUCTION – RNA SYNTHESIS
Transcription is, chemically and enzymatically, very similar to DNA replication. Both involve enzymes that synthesize a new strand of nucleic acid complementary to a DNA template strand. There are some important differences, of course; most notably, in the case of transcription, the new strand is made from ribonucleotides rather than deoxyribonucleotides. Other mechanistic features of transcription that differ from that of replication include the following.
RNA polymerase (the enzyme that catalyzes RNA synthesis) does not need a primer; rather, it can initiate transcription de novo (although in vivo, initiation is permitted only at certain sequences.
Transcription, although very accurate, is less accurate than replication (one mistake occurs in 10,000 nucleotides added, compared with one in 10 million for replication). This difference reflects the lack of extensive proofreading mechanisms for transcription, although two forms of proofreading for RNA synthesis do exist.
During replication the entire chromosome is usually copied, but transcription is more selective. Only particular genes or groups of genes are transcribed at any one time, and some portions of the DNA genome are never transcribed. The cell restricts the expression of genetic information to the formation of gene products needed at any particular moment.
Specific regulatory sequences mark the beginning and end of the DNA segments to be transcribed and designate which strand in duplex DNA is to be used as the template. The transcript itself may interact with other RNA molecules as part of the overall regulatory program.
Initiation of RNA synthesis at random points in a DNA molecule would be an extraordinarily wasteful process. Instead, an RNA polymerase binds to specific sequences in the DNA called promoters, which direct the transcription of adjacent segments of DNA (genes). The sequences where RNA polymerases bind are variable, and much research has focused on identifying the particular sequences that are critical to promoter function.
In E. coli, RNA polymerase binding occurs within a region stretching from about 70 bp before the transcription start site to about 30 bp beyond it. By convention, the DNA base pairs that correspond to the beginning of an RNA molecule are given positive numbers, and those preceding the RNA start site are given negative numbers.
The promoter region thus extends between positions σ 70 and +30. Analyses and comparisons of the most common class of bacterial promoters (those recognized by an RNA polymerase holoenzyme containing σ 70 ) have revealed similarities in two short sequences centered about positions −10 and −35. These sequences are important interaction sites for the σ 70 subunit.
Although the sequences are not identical for all bacterial promoters in this class, certain nucleotides that are particularly common at each position form a consensus sequence (recall the E. coli oriC consensus sequence. The consensus sequence at the −10 region is (5′)TATAAT(3′); at the −35 region it is (5′)TTGACA(3′). A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions −40 and −60 in the promoters of certain highly expressed genes.
The UP element is bound by the α subunit of RNA polymerase. The efficiency with which an RNA polymerase containing σ 70 binds to a promoter and initiates transcription is determined in large measure by these sequences, the spacing between them, and their distance from the transcription start site.
- RNA polymerase requires DNA for activity and is most active when bound to a double-stranded DNA.
- RNA polymerase holoenzyme. The RNA polymerase holoenzyme of E. coli thus exists in several forms, depending on the type of σ subunit. The most common subunit is σ 70 (Mr 70,000).
- The several subunits of the bacterial RNA polymerase give the enzyme the shape of a crab claw. The pincers are formed by the large β and β′ subunits.
PROCESS OF RNA SYNTHESIS
- Initiation of transcription describes the formation of first phosphodiester bond between nucleotide in RNA.
- The initial binding of RNA polymerase to the promoter DNA in the closed complex leaves the DNA in double-stranded form. Refer to as closed binary complex. Closed means that DNA is in duplex form.
- The next stage in initiation requires the enzyme to become more intimately engaged with the promoter, sigma factor changes the ability of DNA binding property of RNA polymerase. Now the closed complex is converted into the open complex. Open complex is formed by melting the short region of DNA leading to transcription bubble. The length of transcription bubble is approximately 12 to 14 bp.
- This “melting” occurs between positions –11 and +2, with respect to the transcription start site.
- Formation of the closed complex, in contrast, is readily reversible: polymerase can as easily dissociate from the promoter as make the transition to the open complex.
- The active site of the enzyme, which is made up of regions from both the b and b0 subunits, is found at the base of the pincers within the active center cleft.
- The NTP-uptake channel allows ribonucleotides to enter the active center. The RNA-exit channel allows the growing RNA chain to leave the enzyme as it is synthesized during elongation. The remaining three channels allow DNA entry and exit from the enzyme.
- Ternary complex contains RNA, DNA and enzyme.
- Polymerase manages to escape from the promoter and enter the elongation phase only once it has managed to synthesize a transcript of a threshold length of 10 or more nucleotides. Once this length, the transcript cannot be accommodated within the region where it hybridizes to the DNA and must start threading into the RNA exit channel. Promoter escape is associated with the breaking of all interactions between polymerase and promoter elements and between polymerase and any regulatory proteins operating at the given promoter.
- During elongation polymerase uses a step mechanism: using single-molecule techniques, it was shown that the enzyme steps forward as a molecular motor, advancing in a single step a distance equivalent to a base pair for every nucleotide it adds to the growing RNA chain. In addition, the size of the bubble, that is, the length of DNA that is not double-helical, remains constant, throughout elongation: as 1 bp is separated ahead of the processing enzyme, 1 bp is formed behind it.
- As well as synthesizing the transcript, RNA polymerase performs two proofreading functions on that growing transcript. The first of these is called pyrophosphorolytic editing. In this, the enzyme uses its active site, in a simple back-reaction, to catalyze the removal of an incorrectly inserted ribonucleotide, by reincorporation of PPi. The enzyme can then incorporate another ribonucleotide in its place in the growing RNA chain. Note that the enzyme can remove either correct or incorrect bases in this manner, but spends longer hovering over mismatches than matches, and thus removes the former more frequently.
- In the second proofreading mechanism, called hydrolytic editing, the polymerase backtracks by one or more nucleotides and cleaves the RNA product, removing the error-containing sequence.
RNA synthesis is processive; that is, the RNA polymerase introduces a large number of nucleotides into a growing RNA molecule before dissociating. This is necessary because, if the polymerase released an RNA transcript prematurely, it could not resume synthesis of the same RNA and would have to start again. However, an encounter with certain DNA sequences results in a pause in RNA synthesis, and at some of these sequences transcription is terminated. Our focus here is again on the well-studied systems in bacteria. E. coli has at least two classes of termination signals: one class relies on a protein factor called ρ (rho), and the other is ρ-independent.
Most ρ-independent terminators have two distinguishing features. The first is a region that produces an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand. The second feature is a highly conserved string of three A residues in the template strand that are transcribed into U residues near the 3′ end of the hairpin.
When a polymerase arrives at a termination site with this structure, it pauses. Formation of the hairpin structure in the RNA disrupts several A=U base pairs in the RNA-DNA hybrid segment and may disrupt important interactions between RNA and the RNA polymerase, facilitating dissociation of the transcript.
The ρ-dependent terminators lack the sequence of repeated A residues in the template strand but usually include a CA-rich sequence called a rut (rho utilization) element. The ρ protein associates with the RNA at specific binding sites and migrates in the 5′ → 3′ direction until it reaches the transcription complex that is paused at a termination site. Here it contributes to release of the RNA transcript.
The ρ protein has an ATPdependent RNA-DNA helicase activity that promotes translocation of the protein along the RNA, and ATP is hydrolyzed by the ρ protein during the termination process. The detailed mechanism by which the protein promotes the release of the RNA transcript is not known.
- Gene expression is the process by which the information in the DNA double helix is converted into the RNAs and proteins whose activities bestow upon a cell its morphology and functions. Transcription is the first step in gene expression and involves copying DNA into RNA. This process is catalyzed by the enzyme RNA polymerase.
- Transcription is catalyzed by DNA-dependent RNA polymerases, which use ribonucleoside 5′-triphosphates to synthesize RNA complementary to the template strand of duplex DNA. Transcription occurs in several phases: binding of RNA polymerase to a DNA site called a promoter, initiation of transcript synthesis, elongation, and termination.
- Bacterial RNA polymerase requires a special subunit to recognize the promoter. As the first committed step in transcription, binding of RNA polymerase to the promoter and initiation of transcription are closely regulated. Transcription stops at sequences called terminators.
- Not only are different parts of the genome transcribed to different extents, but the choice of which part to transcribe, and how extensively, can also be regulated. Thus, in different cells, or in the same cell at different times, different sets of genes might be transcribed. Therefore, for example, two genetically identical cells in a human will, in many cases, transcribe different sets of genes, leading to differences in the character and function of those two cells (e.g., one might be a muscle cell and the other a neuron). Or a given bacterial cell will transcribe a different set of genes, depending on the medium in which it is growing.
- Transcription selectively copies only certain parts of the genome and makes anywhere from one to several hundred, or even thousand, copies of any given section. In contrast, replication must copy the entire genome and do so once (and only once) every cell division. The choice of which regions to transcribe is not random: there are specific DNA sequences that direct the initiation of transcription at the start of each region and others at the end that terminate transcription.