The below mentioned article provides notes on RNA synthesis.
RNA synthesis, also known as DNA transcription, essentially consists of making an RNA copy of a segment of DNA. In this process, a complementary single-stranded RNA molecule is synthesized using one of the two strands of a DNA segment as template. The strand so copied is known as the coding strand of DNA. The enzyme catalyzing transcription is RNA-polymerase which is a multi-protein complex.
There are three main kinds of RNA, viz. messenger RNA (m-RNA), ribosomal RNA (r-RNA) and transfer RNA (t-RNA), all of which play specific roles in protein synthesis. All the three types of RNAs are synthesized by RNA polymerases. While in prokaryotes, all the three classes of RNA are synthesized by a single RNA polymerase, in eukaryotic organisms there are three different RNA polymerases catalyzing synthesis of different classes of RNA molecules.
In E. coli, RNA polymerase is a multi-protein complex. This core complex consists of four subunits of three types — two a-subunits and one each of β and β’ subunits (αββ’). Several additional proteins become associated to the core polymerase and are dissociated during the different phases of transcription. An important protein of this type is the sigma-factor which is associated with the core RNA polymerase during initiation of RNA synthesis and dissociated when the polynucleotide chain is about 8-nucleotide long.
After dissociation of the sigma factor, other proteins, known as elongation factors, become associated with the RNA polymerase. Another protein factor, known as rho-factor, is often necessary (though not always) for termination of the transcription process. RNA polymerase of E. coli is a large protein molecule. Together with the sigma factor it has a molecular weight of 465,000 Daltons.
RNA molecules are single-stranded, although sometimes the single-strand may fold to form various three-dimensional configurations by intrastrand base-pairing as observed in different transfer RNAs. RNA consists of four types of ribonucleotides viz. AMP, GMP, CMP and UMP.
During transcription, precursors of these ribonucleotides are ATP, GTP, CTP and UTP respectively. Base pairing with the coding strand of DNA is between A=U and G=C, i.e. when a coding strand has a sequence of ….ATTGC…, the transcript (RNA) will have a complementary sequence …UAACG…
… A-T-T-G-C… DNA coding strand
… U-A-A-C-G… RNA strand
It should be noted that the transcript has the same base sequence as that of the non-coding DNA strand, except that, in RNA, TMP is replaced by UMP, and that the nucleotides are ribonucleotides and not deoxyribonucleotides.
A general chemical structure of the precursors in RNA synthesis viz. ribonucleoside 5′-triphosphate is shown below:
The chemical events in RNA synthesis are more or less analogous to those of DNA synthesis, although there are certain important differences.
The chemical aspects of RNA synthesis are discussed below and the differences from DNA synthesis are mentioned:
(i) RNA is synthesized using ribonucleoside 5′-triphosphates as precursors. In DNA the precursors are deoxyribonucleoside 5′-triphosphates. The first precursor to initiate RNA chain elongation remains attached at the 5′-end with all the three phosphates. That is, an RNA molecule always contains a 5′-triphosphate at one end.
Chain elongation takes place by addition of ribonucleotide monophosphates to the 3′-OH group of the preceding ribonucleotide through the formation of a phosphodiester bond. A pyrophosphate group of the precursor is released. Thus, the reaction is analogous to the one occurring in DNA synthesis.
The reaction is shown in Fig. 9.27:
An important difference between RNA and DNA synthesis is that RNA polymerase does not require a primer, whereas DNA polymerase cannot initiate DNA synthesis unless an RNA-primer is present. In RNA synthesis, the first nucleotide to initiate chain elongation is a 5′-triphoshate of a ribonucleoside and the three phosphate groups persist in the completed RNA.
Obviously, the base sequence of RNA is determined by the base sequence of the template which is the coding strand of DNA. Each nucleotide added to the elongating RNA strand is selected on the basis of its ability to effectively base-pair with the nucleotide on the template. Although in the synthesis of any one RNA a particular segment of only one strand of DNA is used as the template, it should be noted that the other strand of DNA may also be utilized for synthesis of another RNA.
This is diagrammatically shown in Fig. 9.28:
(ii) The next and the most crucial step of RNA synthesis is the interaction of the RNA polymerase and the template DNA leading to a tight binding between the two. RNA polymerase with the associated sigma factor binds to the template at a specific site of the DNA molecule, known as the promoter. The promoter has a specific base sequence.
The sigma factor helps in recognition of the promoter sequence and, therefore, its association with the core polymerase is essential for binding of the enzyme to the promoter. RNA polymerase is a large complex molecule, and when it binds to DNA, a wide area of the DNA containing many bases is covered by it. This portion of the DNA is protected by the RNA polymerase from the action of DNase.
The protected portion includes the promoter which contains a start site where RNA synthesis begins i.e. the DNA base which pairs with the first RNA base. This site is conventionally indicated as +1. The DNA bases following the start site in the direction of transcription are designated as downstream sequences and they are given a ‘+’ sign. On the other hand, the bases situated in the opposite direction from the start site are known as upstream sequences and indicated by ‘-‘ sign.
The promoter region includes, besides the start site, two other short upstream sequences, which have been shown to be important in recognition of the promoter by the RNA-polymerase. These short sequences are situated at about -10 and -35 upstream positions. The sequence nearer to the start site, i.e. at -10 position, is known as Pribnow box.
It contains the consensus sequence 5T-A-T-A-T-T3′ or 5T-A-T-A-A-T3′ in the non-coding DNA strand. The last base of the sequence stands at a distance of 5 to 6 bases from the start-site. The other important site at the -35 position has a consensus sequence 5’T-T-G-A -C-A3′ in the non-coding strand of DNA. This site may be important in initial binding of the RNA polymerase to the promoter. The initial binding is strengthened by the sigma-factor.
The binding of RNA polymerase to DNA at the promoter site, strengthened by the sigma factor, results in the formation of what is known as a closed promoter complex, because the DNA strands are still not melted. Following tight binding, the RNA polymerase protein undergoes a conformational change which transforms the closed complex into an open promoter complex.
The DNA double helix in this region is melted to expose single-stranded regions extending over from about -21 position to +20 position including the Pribnow box.
A closed and an open promoter complex are diagrammatically shown in Fig. 9.29:
(iii) Binding of the RNA polymerase to the promoter site causes unwinding of the DNA double helix. One of the two strands acts as the template and the first 5′-ribonucleotide triphosphate is incorporated by base-pairing with the DNA nucleotide in position +1 of the template strand.
The subsequent ribonucleotides are added one by one at the 3′-OH end of the previous nucleotide with release of a pyrophosphate group at each addition. The polyribonucleotide chain grows in length as the RNA polymerase moves along the template strand downstream.
After about eight nucleotides have been added, the sigma factor leaves the RNA-polymerase to be recycled to another polymerase molecule. The average rate of polymerization in RNA synthesis in E. coli is about 60 nucleotides per second. RNA polymerase moves along the template strand in the 5′ —> 3′ direction till it reaches the termination signal. The enzyme then releases the newly synthesized RNA molecule and it itself leaves the template.
During the elongation phase, the newly synthesized RNA remains bound to the template DNA for a short distance forming a heteroduplex (DNA-RNA). As the RNA chain elongates, the 5′-end is displaced from the template forming a branch. The portion of the template that has already been transcribed is re-associated with the non-coding strand to reconstitute the double helix. The regeneration of the DNA is also a function of the RNA polymerase.
The sequence of events in RNA synthesis is presented in Fig. 9.30:
(iv) Termination of RNA synthesis takes place by two different mechanisms. One is called self- termination and the other requires the rho-factor and is known as rho-induced termination. Self-termination is caused by the presence of a special base sequence in the template strand of DNA. This sequence contains a length of inverted repeats with an intercalary non-repeating sequence.
The presence of the repeating and non-repeating sequences results in the formation of a stem and loop towards the end of the RNA molecule which is believed to halt RNA synthesis.
In E. coli, the non- coding strand of DNA and the RNA synthesized against the template strand which is used as termination signal are shown below:
Due to the presence of the repeating and non-repeating sequences, the RNA forms a stem and loop structure as shown in Fig. 9.31. The E. coli RNA polymerase stops when it synthesis a self- complementary strand with a loop and a sequence of U’s at the 3′-OH end of the RNA molecule. The base-sequence in the inverted repeats may vary widely in different organisms.
The mechanism of rho-mediated termination of RNA synthesis is not clearly understood. One hypothesis is that during RNA polymerization when a sequence rich in C is synthesized, the rho-factor binds to the RNA polynucleotide chain and prevents the nucleoside triphosphate precursors to reach the RNA polymerase. The rho-protein possesses ATPase activity and the rho-protein degrades the triphosphate and thereby prevents RNA chain elongation. Eventually, the RNA chain dissociates from the template and RNA is released.
(v) Eukaryotic RNA synthesis:
DNA transcription is based mainly on the knowledge derived from the studies in bacteria, specially E. coli. RNA synthesis in eukaryotic cells essentially follows the same pattern, but is more complex in details.
The important differences between the prokaryotic and eukaryotic transcription are briefly discussed below:
In contrast to prokaryotes, which have a single RNA-polymerase catalyzing synthesis of all types of RNAs, the eukaryotes — starting from yeasts to mammals — have three RNA polymerases. Polymerase I makes only ribosomal RNAs. Polymerase II synthesizes, m-RNA and polymerase III produces transfer-RNAs and also 5S r-RNA. The eukaryotic RNA-polymerases are larger in size than the prokaryotic counterparts and they are more complex, containing 10 or more subunits.
The eukaryotic polymerases are located in the cell nucleus where they catalyse transcription. After synthesis, the RNA molecules are processed and these are then trans-located through nuclear membrane pores into the cytoplasm. In prokaryotic cells, the membrane-bound nucleus is absent and the RNAs are released directly in the cell cytoplasm.
A characteristic feature of the eukaryotic genetic material — specially of the mammals — is that a relatively small proportion of the total DNA present in the cell is used to produce RNA of different kinds. Moreover, the RNA molecules transcribed from the DNA, known as the primary transcript, have to be processed to remove a considerable portion to produce the functional RNA molecules.
The portions thus removed may be up to 90% of the primary transcript. In case of m-RNAs, the discarded segments are known as introns and the segments which are retained in the functional molecules as exons. The excision of the introns is achieved by RNA-splicing. The primary transcript containing both introns and exons in case of eukaryotes is also known as heterogeneous nuclear RNA (hn-RNA).
A major difference between prokaryotic and eukaryotic m-RNA is that the eukaryotic hn-RNA is modified at the 5′ and 3′ ends by capping and tailing, respectively. The 5′-(P)-(P)-(P) end of the growing RNA-polynucleotide chain of the primary transcript is capped when the chain is about 30 nucleotide long by 7-methylguanosine coming from GTP.
In the process, the three phosphate groups of the 5′ terminal ribonucleotide are released as Pi and pyrophosphate. The guanosine is then methylated to form the 5′-cap. This capping is necessary to protect the primary transcript from degradation by RNase. The primary transcripts of most eukaryotes contain a poly-A tail consisting of 100-200 adenylic acid residues. The addition of poly-A tail is catalysed not by RNA polymerase, but a different enzyme, poly- A polymerase.
The poly-A tail serves two functions. Firstly, it helps in transporting the m-RNA from the nucleus to the cytoplasm. Secondly, it serves as a recognition signal for the ribosome. The poly-A tail is added after the growing RNA chain is cleaved. The cleavage site is signaled by an RNA sequence AAUAA located 10-25 bases upstream from the 3′-end.
A further difference between prokaryotic and eukaryotic m-RNAs is that prokaryotic m-RNAs are generally polycistronic, while those of eukaryotes are monocistronic. This means that, in prokaryotes, several contiguous cistrons (segments of DNA encoding individual polypeptides) are transcribed together to form a long polycistronic m-RNA. These long polynucleotide chains are later cleaved into monocistronic units before they are attached to ribosomes for translation into proteins.
Another significant feature of the eukaryotic RNA synthesis which is not observed in prokaryotes is that the primary transcript, almost immediately after its synthesis is completed, is coated with proteins to form a string of particles. These are known as heterogeneous nuclear ribonucleoprotein particles (hn-RNP).