About DNA Transcription
Transcription is the synthesis of a single-stranded RNA copy of a segment of DNA. In the case of protein synthesis, a protein-coding gene is transcribed to give a messenger RNA.
Translation (protein synthesis) is the conversion of the messenger RNA base-sequence information into the amino acid sequence of a polypeptide. Transcription is the process of transferring the genetic information in DNA into RNA base sequences.
The DNA unwinds in a short region next to a gene, and an RNA polymerase catalyzes the synthesis of an RNA molecule in the 5′-to-3′ direction along the 3′-to-5′ template strand of the DNA. Only one strand of the double stranded DNA is transcribed into an RNA molecule.
The production of an RNA by transcription of a gene is one step of gene expression. There are four main types of RNA molecules, each encoded by its own type of gene, but only one of them is translated:
1. mRNA (messenger RNA) encodes the amino acid sequence of a polypeptide. mRNAs are the transcripts of protein-coding genes. Translation of an mRNA produces a polypeptide.
2. rRNA (ribosomal RNA), with ribosomal proteins, makes up the ribosomes—the structures on which mRNA is translated.
3. tRNA (transfer RNA) brings amino acids to ribosomes during translation.
4. snRNA (small nuclear RNA), with proteins, forms complexes that are used in eukaryotic RNA processing to produce functional mRNAs.
How is an RNA chain synthesized?
Associated with each gene are sequences called gene regulatory elements, which are involved in regulating transcription. The enzyme RNA polymerase catalyses the process of transcription. (More rigorously, the enzyme is known as DNA-dependent RNA polymerase because it uses a DNA template for the synthesis of an RNA chain.)
The DNA double helix unwinds for a short region next to the gene before transcription begins. In bacteria, RNA polymerase is responsible for unwinding; in eukaryotes, unwinding is done by other proteins that bind to the DNA near the start point for transcription.
In transcription, RNA is synthesized in the 5′-to-3′ direction. The 3′-to-5′ DNA strand that is read to make the RNA strand is called the template strand. The 5′-to-3′ DNA strand complementary to the template strand, and having the same polarity as the resulting RNA strand, is called the non-template strand.
By convention, in the literature and databases of gene sequences, the sequence presented is of the non-template DNA strand. From this strand, the sequence of the RNA transcript can be directly derived and, if it is an mRNA, the encoded amino acids can be directly read from the genetic code dictionary.
The RNA precursors for transcription are the ribonucleoside triphosphates ATP, GTP, CTP, and UTP, collectively called NTPs (nucleoside triphosphates).
RNA synthesis occurs by polymerization reactions similar to those involved in DNA synthesis. RNA polymerase selects the next nucleotide to be added to the chain by its ability to pair with the exposed base on the DNA template strand.
Unlike DNA polymerases, RNA polymerases can initiate new RNA chains; in other words, no primer is needed. Recall that RNA chains contain nucleotides with the base uracil instead of thymine and that uracil pairs with adenine.
Therefore, where an A nucleotide occurs on the DNA template chain, a U nucleotide is placed in the RNA chain instead of a T. For example, if the template DNA strand reads 3′-ATACTGGAC-5′ then the RNA chain will be synthesized in the 5′-to-3′ direction and will have the sequence 5′-UAUGACCUG-3′.
Steps of Transcription
The transcription process is held in 3 steps viz. Initiation of Transcription at Promoters, Elongation of an RNA Chain, Termination of an RNA Chain.
Initiation of Transcription at Promoters
- A bacterial gene may be divided into three sequences with respect to its transcription:
- A promoter, a sequence upstream of the start of the gene that encodes the RNA. The RNA polymerase interacts with the promoter.
- The way the RNA polymerase interacts, spatially speaking, defines the direction for transcription and, thus, dictates to the enzyme which DNA strand is the template strand and where transcription is to begin.
- That is, the Gene Expression: Transcription promoter sequence serves to orient the RNA polymerase to start transcribing at the beginning of the gene and ensures that the initiation of synthesis of every RNA occurs at the same site.
- A gene with its promoter is an independent unit. This means that the strand of the double helix that is the template strand is gene specific. In other words, some genes use one strand of the DNA as the template strand, while other genes use the other strand. The present organization of genes in this regard is the result of the evolution of present-day genomes.
- The RNA-coding sequence itself—that is, the DNA sequence transcribed by RNA polymerase into the RNA transcript.
- A terminator, specifying where transcription stops.
From comparisons of sequences upstream of coding sequences and from studies of the effects of specific base-pair mutations at every position upstream of transcription initiation sites, two DNA sequences in most promoters of E. coli genes have been shown to be critical for specifying the initiation of transcription. These sequences generally are found at-35 and-10, that is, at 35 and 10 base pairs upstream from the+1 base pair at which transcription starts. The consensus sequence (the base found most frequently at each position) for the-35 region (the-35 box) is 5’-TTGACA-3’. The consensus sequence for the-10 region (the 10 box, formerly called the Pribnow box, after David Pribnow, the researcher who first discovered it) is 5’-TATAAT-3’.
Only one type of RNA polymerase is found in bacteria, so all classes of genes—protein-coding genes, tRNA genes, and rRNA genes—are transcribed by it. Initiation of transcription of a gene requires a form of RNA polymerase called the holoenzyme (or complete enzyme).
The holoenzyme consists of the core enzyme form of RNA polymerase, which consists of two , one , and one polypeptide, bound to another polypeptide called a sigma factor.
The sigma factor ensures that the RNA polymerase binds in a stable way only at promoters. That is, without the sigma factor, the core enzyme can bind to any sequence of DNA and initiate RNA synthesis, but this transcription initiation is not at the correct sites.
The association of the sigma factor with the core enzyme greatly reduces the ability of the enzyme to bind to DNA non-specifically and establishes the promoter-specific binding properties of the holoenzyme. A sigma factor is not required for the elongation and termination stages of transcription. The RNA polymerase holoenzyme binds to the promoters of most genes as shown in.
First, the holoenzyme contacts the-35 sequence and then binds to the full promoter while the DNA is still in standard double helix form, a state called the closed promoter complex. Then the holoenzyme untwists the DNA in the-10 region. The untwisted form of the promoter is called the open promoter complex. The sigma factor of the holoenzyme plays a key role in these steps by contacting the promoter directly at the-35 and-10 sequences.
Once the RNA polymerase is bound at the10 box, it is oriented properly to begin transcription at the correct nucleotide of the gene. At this point the RNA polymerase is contacting about 75 bp of the DNA from-55 to 120
Promoters differ in their sequences, so the binding efficiency of RNA polymerase varies. As a result, the rate at which transcription is initiated varies from gene to gene. For example, a-10 region sequence of 5’-GATACT-3’ has a lower rate of transcription initiation than does 5’-TATAAT-3’ because the ability of the sigma factor component of the RNA polymerase holoenzyme to recognize and bind to the first sequence is lower than it is to the second sequence.
As already mentioned, the promoters of most genes in E. coli have the-35 and-10 recognition sequences. Those promoters are recognized by a sigma factor with a molecular weight of 70,000 Da, called 70. There are other sigma factors in E. coli with important roles in regulating gene expression
Each type of sigma factor binds to the core RNA polymerase and permits the holoenzyme to recognize different promoters. For example, under conditions of high heat (heat shock) and other forms of stress, 32 (molecular weight 32,000 Da) increases in amount, directing some RNA polymerase molecules to bind to the promoters of genes that encode proteins needed to cope with the stress. Such promoters have consensus recognition sequences specific to the 32 factor at-39 and-15.
There are several other types of sigma factors with various roles. In brief, the transcription of many bacterial genes is controlled by the interaction of regulatory proteins with regulatory sequences upstream of the RNA-coding sequence in the vicinity of the promoter.
There are two classes of regulatory proteins: activators stimulate transcription by making it easier for RNA polymerase to bind or elongate an RNA strand, while repressors inhibit transcription by making it more difficult for RNA polymerase to bind or elongate an RNA strand.
Elongation of an RNA Chain
- RNA synthesis takes place in a region of DNA that has separated into single strands to form a transcription bubble.
- Once initiation get ahead and the elongation stage is recognized, the RNA polymerase begins to move along the DNA and the sigma factor is released.
- The core enzyme alone is able to complete the transcription of the gene. In E. coli growing at 37°C, transcription occurs at about 40 nucleotides/sec. During the transition from initiation to elongation, the RNA polymerase becomes more compact, contacting less of the DNA.
- Once the elongation stage is established, the RNA polymerase contacts about 40 bp of the DNA with approximately 25 bp in the transcription bubble.
- During the elongation stage, the core RNA polymerase moves along, untwisting the DNA double helix ahead of itself to expose a new segment of single-stranded template DNA.
- Behind the untwisted region, the two DNA strands reform into double-stranded DNA. Within the untwisted region, about 9 RNA nucleotides are base-paired to the DNA in a temporary RNA–DNA hybrid; the rest of the newly synthesized RNA exits the enzyme as a single strand.
- RNA polymerase has two proofreading activities. One of these is similar to the proofreading by DNA polymerase, in which the incorrectly inserted nucleotide is removed by the enzyme reversing its synthesis reaction, backing up one step, and then replacing the incorrect nucleotide with the correct one in a forward step.
- In the other proofreading process, the enzyme moves back one or more nucleotides and then cleaves the RNA at that position before resuming RNA synthesis in the forward direction.
Termination of an RNA Chain
- The termination of bacterial gene transcription is signaled by terminator sequences.
- In E. coli, the protein Rho (ᵨ) plays a role in the termination of transcription of some genes. The terminators of such genes are called Rho-dependent terminators (also, type II terminators). For other genes, the core RNA polymerase terminates transcription; terminators for those genes are called Rho-independent terminators (also, type I terminators).
- Rho-independent terminators consist of an inverted repeat sequence that is about 16 to 20 base pairs upstream of the transcription termination point, followed by a string of about 4 to 8 A–T base pairs.
- The RNA polymerase transcribes the terminator sequence, which is part of the initial RNA-coding sequence of the gene. Because of the inverted repeat arrangement, the RNA folds into a hairpin loop structure. The hairpin structure causes the RNA polymerase to slow and then pause in its catalysis of RNA synthesis.
- The string of U nucleotides downstream of the hairpin destabilizes the pairing between the new RNA chain and the DNA template strand, and RNA polymerase dissociates from the template; transcription has terminated.
- Mutations that disrupt the hairpin partially or completely prevent termination.
- Rho-dependent terminators are C-rich, G-poor sequences that have no hairpin structures like those of rho-independent terminators. Termination at these terminators is as follows: Rho binds to the C-rich terminator sequence in the transcript upstream of the transcription termination site.
- Rho then moves along the transcript until it reaches the RNA polymerase, where the most recently synthesized RNA is base paired with the template DNA. Rho is a helicase enzyme, meaning that it can unwind double-stranded nucleic acids.
- When Rho reaches the RNA polymerase, helicase unwinds the helix formed between the RNA and the DNA template strand, using ATP hydrolysis to provide the needed energy. The new RNA strand is then released, the DNA double helix reforms, and the RNA polymerase and Rho dissociate from the DNA; transcription has terminated.
References and Sources
- 10 Instruments Used in Microbiology Laboratory
- 8 Qualitative Tests for Protein
- Aberration In Lens System
- Acid Fast Staining
- Aseptic Transfer Technique
- Bacterial Flagella, Fimbriae and Pili
- Bacterial Growth and Nutrition
- Fimbriae vs Flagella
- Fundamental Microscopy
- Growth Curve of Bacteria
- MacConkey agar
- McFarland Standards
- Monochrome Staining
- Negative Staining
- Ninhydrin Test
- Serial Dilution in Microbiology
- Spread Plate Technique
- Streak Plate Technique
- Types of Extremophiles
- Xanthoproteic Test