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Central Dogma of Molecular Biology – Stages of Transcription, RNA Polymerase
The concept of central dogma was first proposed by Francis Crick and the concept says about the flow of information from DNA to protein. Genes are responsible for the expression of phenotypic features. Those are mainly mediated by the production of proteins. A gene is represented by a segment of DNA which is a sequence of nucleotides and the sequence of nucleotides remains at the pivotal position for the formation of a protein when a protein is made up of a sequence of amino acids. Therefore, the sequence of nucleotides of the DNA bears the information for a protein.
In this context, the genetic material, DNA of any species is the storehouse of information for various types of proteins. This information remains in coded language and coded language is translated into amino acid chain or polypeptide in a protein. However, the information in the gene of DNA is transferred as a message in the form of RNA, named messenger RNA (mRNA) which is produced from DNA by a process called transcription. The mRNA then may produce a polypeptide chain by a process called translation. The formation of a polypeptide chain is the initial step in the process of expression of a gene. Hence, Crick proposed that information stored in DNA flows unidirectionally to produce a polypeptide chain for the expression of a phenotypic feature in living organisms. This flow of information may be depicted by the following diagram.
DNA → RNA → Protein
Some modifications over this initial concept were inevitable after the discovery of reverse transcriptase from retroviruses. The enzyme reverse transcriptase promotes the formation of DNA from the RNA genome of the retroviruses in their host cells and with the help of this enzyme, a short DNA copy may be made from an mRNA. Therefore, a reverse flow of information may occur from RNA to DNA. Further, when a DNA molecule replicates forming its replica, the information stored in the DNA may be transferred to its daughter DNA molecules. The present form of Central Dogma has been given before.
Transcription and RNA Synthesis
The gene in the form of DNA may send its message to RNA in the coded form and this may directly participate in the process of gene expression. Hence, the production of RNA from DNA is an important life phenomenon. The process by which RNA molecule is synthesized using DNA as a template, thereby transferring genetic information from DNA to RNA is known as transcription.
In the same way, as replication occurs in the cell, with the use of cellular DNA as a template, RNA polymerase may polymerize ribonucleotides (ATR GTP, CTP & UTP) to form different RNA transcripts. The nascent RNA transcripts in most cases are processed to produce functional molecules.
General Features of Transcription
In all living organisms, transcription occurs on the basis of the following principles :
- Transcription is selective i.e., the entire molecule is not transcribed.
- The cellular DNA acts as a template for RNA synthesis and in the process one of the two strands of the DNA helps in the polymerization of complementary ribonucleotides over it.
- This strand of DNA is known as the template strand, while the other strand of DNA is called the nontemplate or – strand.
- RNA synthesis is a commonly promoter-mediated event when some specific sequences of the DNA act as promoters for RNA synthesis.
- Three stages of transcription: Initiation, Elongation, and termination.
- Elongation of the RNA chain during its synthesis occurs along the 5′ to 3′ direction.
- Products of transcription: Inactive primary RNA transcripts.
- Inactive primary RNA transcripts undergo post-transcriptional modifications that are splicing, terminal base additions, base modifications etc.
Requirements for Transcription
1. DNA Template
Cellular DNA acts as a template for transcription and the process can only occur in the presence of a template DNA. Only one strand from a segment of DNA representing a gene may participate in RNA synthesis and therefore it is called the template strand. The other strand that does not participate in the process and is called the non-template strand.
It is to be pointed out that the template strand usually contains two distinct segments: one that directs the polymerization of the nucleotides over it, is known as downstream sequence and the other prior to this with its involvement in the process is known as an upstream sequence. The nucleotides along the downstream sequence are marked with +1, +2, +3, and so on, whereas the nucleotides along the upstream sequence are marked with -1, -2, -3, and so on. RNA synthesis starts from point +1.
The upstream region of the template strand contains some important sites very much associated with transcription and these are known as promoters. There may be more than one promoter within a single genetic unit or transcription unit. One regulatory unit of transcription in the case of prokaryotic cells is known as an operon and an operon is constituted of the promoter, operator, and structural genes (Re: Schematic plant of Transcription Unit). In the case of eukaryotes, the existence of such an operon is not seen.
2. RNA Polymerase
This is the DNA-dependent polymerizing enzyme for synthesizing RNA. The enzyme has the capability of unwinding DNA strands as well as polymerization of ribonucleotides over the template strand of DNA. In the prokaryotic cells, only one type of RNA polymerase is present and that may carry out the synthesis of different types of RNAs (i.e., mRNA, tRNA & rRNA) in the cell. On the other hand, in eukaryotic cells, there are three types of RNA polymerases: RNA polymerase-I, RNA polymerase-II, and RNA polymerase-III, for the synthesis of rRNA, mRNA, and tRNA respectively. RNA polymerase-I is associated with rRNAs except for 5S rRNA; RNA polymerase- II is associated with the formation of mRNA and RNA polymerase-III is associated with the formation of tRNA and other small RNA molecules.
(a) Prokaryotic RNA Polymerase: Though the structure of the prokaryotic RNA polymerase was presumed to be identical in all the prokaryotic organisms, the structure of the RNA polymerase from Escherichia coli could be characterized fully. The enzyme molecule is quite large in size having a molecular weight of about 390000 Da The molecule is formed of four sub-units namely α, β, β’, and ω. The molecular dimension of these units are 36,500, 151,000, 1,55,000, and 11000 Da respectively.
There are two sub-units and others are present singly in the complex of the sub-units. The complex of these four different sub-units is known as the core enzyme molecule and this may be associated with another protein sub-unit called σ factor and the enzyme complex with the sigma factor is known as the holoenzyme.
The sigma factor measures about 70,000 Da. During the initiation of transcription, sigma factor is needed to be present with the core enzyme and following initiation, the rest part of the transcription may be carried out by the core enzyme part. The sigma factor can recognize the promoter region and promoter binding of RNA pol. may occur with its involvement. Whereas the core enzyme part may carry out the unwinding of DNA and polymerization of nucleotides over the template strand. The composition of RNA polymerase with its sub-units and the functions of the individual sub-units is shown in the table, on the next page.
Sub-units of RNA Polymerase of E. coli and their Functions:
|Sub-unit Enzyme||Number per Complex||Molecular Weight (Mr)||Function|
|Alpha (α)||2||36,500||Not known|
|Beta (β)||1||1,51,000||Formation of phosphodiester bond|
|Beta Prime (β’)||1||1,55,000||Formation of template bond|
|Omega (ω)||1||11,000||Not known|
|Sigma (σ)||1||70,000||Promoter recognition and initiation of transcription|
(b) RNA Polymerase of Eukaryotic Cells: In eukaryotic cells, three types of RNA polymerase are generally found that differ by their numbers and properties as given in the table.
RNA Polymerases of Eukaryotic Cells and their Properties:
|Enzyme type||Site of location||Associated with the production||Number per cell||Alpha (α) amanitin sensitivity|
|RNA pol. I||Nucleolus||5.8S, 18S & 28S rRNA||15||Not sensitive and remains active|
|RNA pol. II||Nucleoplasm||mRNA & U-snRNA||14||Sensitive and becomes inactive at even low concentration|
|RNA pol. III||Nucleoplasm||tRNA, 5S, rRNA & 7S RNA||17||Inactive at high dose|
All three enzymes are large enough having molecular weight 5,00,000 Dalton to 6,00,000 Dalton. The number of sub-units in each enzyme varies from 14 to 17. Normally each enzyme molecule contains two large sub-units of dissimilar dimensions with molecular weight ranging from 1,20,000 Dalton to 2,20,000 Dalton. Of the other 12 -15 sub-units of the enzyme molecule, sub-units are similar in dimension. There are five additional sub-units in RNA Pol. I, 9 additional sub-units in RNA Pol. II and 8 in RNA Pol. Ill along with six identical sub-units. The large sub-units of the polymerases are comparable to the β and β’ sub-units of E. coli RNA polymerase.
Transcription Factors: In both prokaryotes and eukaryotes there are some factors that may control the process of transcription. In the E. coli cell, CAP or Catabolic gene Activator Protein and cAMP are two such factors. In the absence of these two factors, RNA polymerase cannot carry out its functions. In comparison, eukaryotic cells contain more transcription factors associated with the RNA polymerases to carry out transcription. Several such factors are named as TF-II-D, TF-II-A, TF-II-B, TF- II-F, TF-II-E, TF-II-H and TF-II-J. Besides, some small protein components are also essential for the initiation of transcription such as TBP (TATA Box binding proteins).
During the elongation phase in the eukaryotic system, several enzymes are found to be involved to carry out capping in mRNA synthesis and those are geranyl transferase and 7 methyl guanine transferase, 2-O-methyl transferase. In the transcription termination of eukaryotes, a specificity component having one nuclease and poly A polymerase play a significant role during transcription.
For RNA synthesis, the precursors required are four types of ribonucleotides present in the cellular pool. They are ATP, GTP, CTP, and UTP. During polymerization, they are converted into nucleoside monophosphates with the liberation of two molecules of inorganic pyrophosphates from each of the ribonucleotides. Nucleotides may be combined together with the formation of phosphodiester bonds.
Proteins required for transcription of Eukaryotic structural genes via core promoter:
|TFIID||Comprises of TATA-binding protein (TBP) and other TBP-associated factors (TAFs), TFIID recognizes the TATA box of eukaryotic structural gene promoters.|
|TFIIB||Binds to TFIID and then enables RNA polymerase II to bind to the core promoter apart from promoting TFIIF binding.|
|TFIIR||Binds to RNA polymerase II and plays a role in its ability to bind to TFIIB and the core promoter apart from playing a role in the ability of TFIIE and TFIIH to bind to RNA polymerase II.|
|TFIIE||Plays a role in the formation or the maintenance (or both) of the open complex. It may exert its effects by facilitating the binding of TFIIH to RNA polymerase II and regulating the activity of TFIIH.|
|TFIIH||A multisubunit protein that has multiple roles. First, certain subunits act as helicases and promote the formation of the open complex. Other subunits phosphorylate the carboxyl-terminal domain (CTD) of RNA polymerase II, which releases its interaction with TFIIB, thereby allowing RNA polymerase II to proceed to the elongation phase.|
|TFIIA||Stabilization of TBP binding, stabilization of TAF-DNA binding.|
|TFIIJ||Required for promoter clearance and elongation.|
Mechanism of Transcription
The mechanism of transcription is completed through three events namely Initiation, Elongation, and Termination. The events associated with each of the three steps are known from the studies on E. coli and the mechanism of transcription is basically identical in all living organisms except a few exceptional features in eukaryotes.
Initiation of transcription is completed through three events, namely Binding of RNA polymerase with promoter, Formation of open complex, and Incorporation of first ribonucleotide.
In the upstream region, the transcription unit contains several promoter sequences which may interact with the RNA polymerase before the commencement of RNA chain formation. In E. coli cells two such promoter sequences are known as -10 and -35 sequences. These sequences are centered around the -10 and -35 nucleotides along the upstream region and within these regions, there are some consensus sequences. At the -10 region, the consensus element comprises 5′ TATAAT 3′. -10 sequence of the prokaryotic promoters is also known as Pribnow Box. It is to be noted that these sequences are present on the non-template strand. The -10 and -35 sequences are separated by a region of about 16 to 19 nucleotides.
Prior to the initiation of transcription, the RNA polymerase recognizes the promoter and binds with it. In the E. coli cell, the holoenzyme molecule with its sigma factor may bind with the double-stranded DNA along the promoter region, and thereby, a closed promoter complex is formed. Following this, the closed complex turns into an open complex when two polynucleotide strands of the DNA get separated. Siebenlist et. al. showed that initially, the RNA polymerase may separate the DNA segment having about 12 base pairs, though the enzyme binds with DNA covering about 70 bp region. After the formation of the open complex, the incorporation of the first nucleotide occurs.
From the -10 region, the first nucleotide incorporation occurs at a site 6 to 7 nucleotides downstream. The first nucleotide incorporated is either PPPA or PPPG. Hawley and McCleore showed that in most of the cases, the sequence at -1, +1, and +2 sites of the coding strand or non-template strand is CAT. Because of this reason, the first two nucleotides incorporated in the transcript are 5’PPPAPU3’. With the incorporation of ribonucleotide at the site of initiation, the open complex is converted into a ternary complex. During this phase after the incorporation of 8 to 9 nucleotides in the RNA chain, the sigma factor is released from the holoenzyme.
From the analysis of the Lac operon of E. coli, it could be revealed that RNA polymerase activity depends on another reaction. The promoter region of this operon may be divisible into two segments RNA polymerase interacting site and the CAP site. In the CAP site, a complex of cAMP-CAP when binds, the RNA polymerase binding at the RNA polymerase interacting site may occur properly and transcription may be initiated.
Following the release of the sigma factor from the RNA polymerase holoenzyme, the phase of elongation starts. In this phase, a gradual and progressive movement of the RNA polymerase core enzyme may be observed. At every step RNA polymerase may move 70 nucleotides forward and under the influence of the enzyme about 17 base pair region shows unwinding and denaturation of strands. At the same time, RNA polymerase also carries out incorporation as well as polymerization of complementary nucleotides at the 3′ OH end of the initially incorporated nucleotides during the initiation phase. This sort of incorporation and polymerization of bases goes on until and unless a termination signal is encountered by the RNA polymerase.
Four types of ribonucleotides, e.g., UTP, CTR GTP, and ATP, participate in the polymerization reaction. It is to be mentioned here that the first nucleotide incorporated is the triphosphonucleoside, while the others during the formation of phosphodiester bonds are converted into nucleoside monophosphates. The transcription bubble formed during the separation of strands moves forwardly along with the incorporation of nucleotides along the separated DNA strand and at this condition reannealing of the strands prior to the new transcription bubble occurs with the release of the RNA chain from the hybridized region. In E. coli, chain elongation occurs at a speed of about 50 nucleotides per second.
As the RNA polymerase reaches the terminal end of a gene under transcription with the final incorporation of the nucleotide at the terminal end, the phase of termination starts. At this phase, both the RNA polymerase and the RNA transcript are detached from the DNA. From the studies on E. coli, it could be revealed that termination of transcription may be accomplished by two different modes, one occurs with the involvement of some termination factors and the other is independent of a termination factor. The factor promoting transcription termination is known as ρ.
In the case of ρ independent termination, transcription may be terminated by some termination signals. The termination signal comprises a GC-rich repeats followed by several adenylates. In between GC-rich regions, a unique sequence is present and the transcript formed from it may form a hairpin loop. As a result, no DNA-RNA hybrid may be formed at the termination site and the RNA polymerase is prevented from moving forwardly. Besides the U residues formed terminally of the transcript pair with the A residues on the template strand. This pairing is very unstable leading to easy separation of the nascent RNA chain from the DNA strand.
In rho-dependent termination, termination of synthesis may be accomplished by the protein factor rho (ρ) which is a multimeric protein with six sub-units having a mol. wt. about 46,000 Da. The rho factor may bind with the 5′ end of the released portion of the nascent RNA chain and then moves along it toward the 3′ end. As it reaches the termination region with RNA polymerase, it influences the activity of the enzyme to be slowed down or stopped. Following this it may take out the RNA end from the DNA strands releasing it in the cytosol.
|Processing||Cleaving of a large RNA transcript into smaller pieces. One or more of the smaller pieces becomes a functional RNA molecule. Processing occurs in the case of rRNA and tRNA transcripts.||Occurs in both prokaryotes and eukaryotes.|
|Splicing||Splicing involves both cleavage and joining of RNA molecules. RNA is cleaved at two sites that allow an internal segment of RNA called intron, to be removed. After the intron is removed, the two ends of the RNA molecules are joined together.||Splicing is common among eukaryotic prem- RNAs and it also occurs occasionally in rRNAs, tRNAs, and a few bacterial RNAs.|
|5′ capping||Attachment of a 7-methylgunsoine cap (m7G) to the 5′ end of mRNA. The cap plays a role in the splicing of introns, exit of mRNA from the nucleus, and binding of mRNA to ribosome.||In eukaryotic mRNAs.|
|3′ polyA tailing||Attachment of a string of adenine-rich nucleotides of 3′ end of mRNA at a site where the mRNA is cleaved. It is necessary for RNA stability and translation in eukaryotes.||In eukaryotic mRNAs and occasionally occurs in bacterial RNAs.|
|RNA editing||Change in the base sequence of an RNA after it has been transcribed.||Occurs occasionally in eukaryotic RNAs.|
|Base modifications||Covalent modification of a base within an RNA molecule.||Base modification commonly occurs in tRNA molecules in both prokaryotes and eukaryotes.|
Temin and Baltimore showed that in some viruses genetic material is RNA and they also contain certain enzymes called reverse transcriptase. This enzyme may use RNA as the template and produces DNA. The RNA virus containing reverse transcriptase is called retrovirus. The mechanism by which DNA is synthesized by the retroviral RNA is known as reverse transcription. The concept of reverse transcription has modified the concept of central dogma originally as proposed by Crick.
During infection, the retroviral RNA is introduced in the cell by these viruses and the viral genome with the help of reverse transcriptase produces a copy of DNA. This DNA is integrated into the host genome (DNA). Under favourable conditions, this DNA part is integrated into the host genome and may become active and produce many copies of the viral RNA.
Cancer-causing retroviruses have been found in man and they are known as tumour RNA viruses. They carry cancer-causing oncogenes. HIV-causing AIDS in men is also one type of retrovirus. In the case of some bacteriophages e.g., f2, MS2, R17, and QB are having RNA genomes. They all can infect E. coli cells. Replication of this viral RNA is dependent on RNA replicase or RNA-dependent RNA polymerase. RNA replicase cannot carry out the replication of RNA of the host. It can only carry out the replication of viral RNA. This replicase is produced in the host cell by the influence of the viral replicase gene.
Difference between prokaryotic and eukaryotic transcription:
Prokaryotic and eukaryotic transcription is basically the same and in both cases the end product is RNA. In both cases, the process is catalyzed by RNA polymerase. In spite of this, there are some fundamental differences. Those may be discussed as under.
|Basis of difference||Prokaryotic transcription||Eukaryotic transcription|
|Mode of occurrence||Transcription and translation occur simultaneously.||Transcription and translation occur separately.|
|Site||It occurs in the cytoplasm.||It occurs in the nucleus.|
|Processing of RNA||RNA is processed in the cytoplasm.||RNA is processed in the nucleus.|
|Initiation factor||It does not require any factor for initiation.||Protein factors are required for transcription initiation.|