Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND REALIZATION OF GENETIC INFORMATION

CHAPTER 20. MOLECULAR MECHANISMS OF DNA REPLICATION AND RNA TRANSCRIPTION

20.4. ENZYMES AND MECHANISMS OF RNA TRANSCRIPTION

The Biosynthesis of RNA on a DNA template is called METABOLISM/31.html">Transcription (from English transcription — copying). The GENERAL PATTERNS OF this process are similar in PROKARYOTES AND EUKARYOTES.

The sequence of incorporation of ribonucleotides into the polyribonucleotide chain during transcription is programmed by The nucleotide sequence in the template DNA. A distinction is made between the coding DNA strand — that is, the one from which Genetic information is read, and the non-coding strand. In double-stranded DNA containing many genes, either of the two strands can serve as the coding strand for some genes, while for other genes, the opposite strand serves this purpose:

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Transcription of various classes of RNA on a DNA template is catalyzed by DNA-dependent RNA polymerases (RNA polymerases), which differ between prokaryotic and eukaryotic organisms. Unlike DNA polymerases, RNA polymerases are capable of independently initiating the synthesis of a polynucleotide chain by binding to specific regions (sites) on the template DNA.

Enzymes and Mechanisms of Transcription in Prokaryotes

In Prokaryotic Cells, unlike Eukaryotic cells (see below), the sole enzyme responsible for synthesizing all three classes of RNA (messenger, transfer, and ribosomal) is DNA-dependent RNA polymerase (RNA polymerase).

E. coli RNA polymerase (molecular weight 465 kDa) is an oligomeric protein composed of several types of subunits: two α-subunits, one β-subunit, and one β'-subunit. These four protomers form the so-called core enzyme (from English core) α₂ββ’, which forms the complete holoenzyme upon interaction with an additional σ-(sigma) subunit that reversibly binds to the α₂ββ’ complex and dissociates at specific Stages of Transcription.

The transcription system in E. coli requires RNA polymerase (holoenzyme), four nucleoside triphosphates (ATP, GTP, UTP, CTP), and a DNA template. The general scheme of the RNA polymerase reaction can be represented by the following equation:

The direction of polyribonucleotide chain synthesis is 5'→3'. Similar to DNA polymerase reactions, the RNA polymerase reaction proceeds via a nucleophilic attack of the 3'-OH group of the terminal ribose of the polyribonucleotide chain on the α-phosphate of the incoming nucleoside triphosphate during chain elongation. The catalytic site that forms the 3'-5'-phosphodiester bonds between ribonucleoside monophosphates is located on the β-subunit of RNA polymerase.

Stages of RNA Synthesis in Prokaryotes

a) binding of RNA polymerase to the DNA template;

b) initiation of polyribonucleotide chain synthesis;

c) elongation (extension) of RNA synthesis;

d) termination, i.e., the completion of RNA synthesis (the so-called "primary RNA transcript").

Interaction of RNA Polymerase with the DNA Template and Transcription initiation

Transcription Promoters

The binding of RNA polymerase to the DNA template occurs at specific Regions of the genome known as promoters. This interaction is mediated by the σ-subunit of RNA polymerase, which subsequently (following the initiation of polyribonucleotide synthesis) dissociates from the core enzyme. Bacterial cells produce a significant number of σ-factors required to initiate the synthesis of various RNAs.

Promoter regions of DNA are sequences approximately 40 NUCLEOTIDES long. The following short (6-nucleotide) sequences found within various promoters are essential for transcription initiation (Fig. 20.12):

Fig. 20.12. Structure diagram of the DNA promoter region.

(1) "-35 sequence" — the TTGACA sequence located 35 nucleotides "upstream" (toward the 5'-end) from the initiation point (the "0 point"), which is believed to be the region interacting with the σ-factor of the RNA polymerase holoenzyme;

(2) "-10 sequence", or the "Pribnow box" — the TATAAT sequence located 10 nucleotides "upstream" from the initiation point. The unwinding of the two DNA strands takes place within the Pribnow box region, making the coding strand accessible to the catalytic sites of RNA polymerase.

Transcription Initiation

Directly, the initiation of RNA synthesis begins with the incorporation of the first (5'-terminal) nucleotide into the chain, which in all mRNAs (in both prokaryotic and eukaryotic organisms) is a purine nucleoside triphosphate (Pur-5'-PPP):

The rate of the initiation process depends on The structure of the promoter sequences preceding the initiation sites. There are strong and weak transcription promoters:

strong promoters drive the initiation of Transcription of the corresponding RNAs at a frequency of up to one initiation event per second;

weak promoters drive transcription initiation at a frequency of about one synthesis start event every 10 minutes.

The efficiency of promoter action depends on The nucleotide sequences located between the "-35" and "-10" blocks; furthermore, promoter activity decreases significantly in the event of Mutations within these promoters.

The interaction of RNA polymerase with the DNA template is blocked by the antitumor antibiotic actinomycin D, which intercalates into the gaps between adjacent Base Pairs, predominantly between G-C pairs (the intercalation process), thereby preventing the enzyme from binding to the polydeoxyribonucleotide chain.

Initiation inhibitors include the antibacterial Antibiotics rifamycin and rifampicin, which block the binding of the first NTPs to the active centers of the RNA polymerase β-subunit.

Transcription Elongation and Termination

As already noted, the elongation of RNA synthesis proceeds in the 5'→3' direction, antiparallel to the coding (template) DNA strand:

During elongation, regions of the double-helical DNA preceding the RNA polymerase are unwound, and 3'-5' phosphodiester bonds are formed within the polyribonucleotide chain. The Selection of the next NMP in the synthesized RNA chain is determined by the STRUCTURE OF THE complementary dNMP in the coding DNA strand, such that the nitrogenous bases A, G, T, C of the DNA chain direct the incorporation of the U, C, A, and G bases into the RNA chain, respectively (Fig. 20.13).

Fig. 20.13. Sequential copying of the coding DNA strand during transcription (Yu.A. Ovchinnikov, 1987).

As a result of elongation, a DNA-RNA hybrid is formed, consisting of the DNA coding strand and the RNA transcript.

Transcription termination occurs under the following conditions:

(1) the RNA polymerase reaches specific terminator regions as it progresses along the DNA template. These terminator regions are characterized by the presence of inverted repeats ("palindromes")*, i.e., nucleotide sequences that read the same in both forward and reverse directions, followed by poly-A-T pairs. The RNA region transcribed from such a palindromic sequence forms a "hairpin" structure followed by a (terminal) UUU... sequence. The formation of RNA with such a structure is a prerequisite for the dissociation of the DNA-RNA hybrid (Fig. 20.14).

Fig. 20.14. The terminator region of a Gene, containing the inverted repeats (TCGGGCG)-(GCGGGCT) and (CGCCCGA)-(AGCCCGC), respectively.

Transcription of the coding strand of the terminator region leads to the formation of an RNA transcript containing complementary sequences (AGCCCGC) and (UCGGGCG) that form hairpin structures.

(2) the auxiliary action of a specific protein terminator, the so-called ρ (rho) factor. The interaction of the ρ-factor with the polymerase complex leads to the dissociation of the DNA-enzyme-RNA complex and the release of the primary transcript.

The sequence of processes constituting DNA-dependent RNA transcription in eukaryotes is shown in Fig. 20.15.

Fig. 20.15. Scheme of the Sequential Stages of transcription involving RNA polymerase (Fer).

Fig. 20.16. Scheme of "hairpin" formation in the RNA transcript. Sequences (1) and (2’) and (2) and (1’) in the DNA strands are pairwise palindromes. The sequences UUACA and UGUAA in the RNA transcript are mutually complementary.

Enzymes and Mechanisms of Transcription in Eukaryotes

RNA Polymerases of Higher Organisms

Eukaryotic cells contain three main classes of RNA polymerases, each responsible for transcribing different sets of genes and synthesizing a specific type of RNA. The Molecular Weight of RNA polymerases from mammalian cells is 500-600 kD.

Table 20.2. Nuclear RNA Polymerases of Animal Cells

In addition to the aforementioned RNA polymerases located in The Nucleus of The Introduction/5.html">Eukaryotic Cell, Mitochondria function with their own RNA polymerase, which ensures the transcription of genes for most mitochondrial Proteins based on the information encoded in the autonomous Mitochondrial Genome.

The nuclear enzyme responsible for transcribing genes that program the synthesis of most cellular proteins is RNA polymerase II, which directs the formation of mRNA precursors—hnRNA—that are heterogeneous in size and nucleotide sequence. RNA polymerase II is specifically inhibited by α-amanitin, a toxin produced by the fungus Amanita phalloides.

Upon the action of RNA polymerase II in eukaryotic cells, monocistronic RNA is formed—that is, RNA which serves as a template for the synthesis of a single polypeptide chain in Ribosomes, in contrast to the polycistronic RNA of prokaryotes.

Transcription Signals

A distinctive feature of RNA synthesis in higher organisms is a more complex system of transcription signals compared to prokaryotes, consisting of specific DNA sequences within the promoter and regulatory proteins that control transcription activity.

The signal for transcription initiation in mammalian genes is the sequence TATAAAAGA, homologous to the Pribnow box (TATAAT), located at position -32 relative to the start point of polyribonucleotide synthesis. RNA polymerase II interacts with this sequence.

The transcription signaling system in eukaryotes also includes signals that not only indicate the initiation site of RNA synthesis but also regulate its activity. These issues will be discussed in more detail in Chapter 23.

Post-transcriptional RNA Modification

In prokaryotic cells, particularly in the most studied bacterial cell—E. coli—mRNA molecules are synthesized immediately in a "mature" form, i.e., ready to perform their biochemical Functions. In contrast, the polyribonucleotide biosynthesis processes occurring in eukaryotic cells yield a primary transcript (heterogeneous nuclear RNA, or pre-mRNA), which is capable of being converted into a functional, complete molecule through post-transcriptional modification reactions known as Processing (maturation).

Primary transcript processing includes:

- attachment of a specific nucleotide structure to the 5'-end of the molecule, the so-called "cap"; the cap is 7-methylguanosine linked via a triphosphate bond to the 5'-terminal nucleotide (Chapter 3, section 3.4);

- attachment of a polyadenylate "tail" to the 3'-end of the primary transcript—a poly(A) sequence 20-250 nucleotides long; The Significance of capping and polyadenylation lies in enhancing the translational activity of mature mRNA and protecting it against the destructive action of cellular ribonucleases;

- excision of non-informative nucleotide sequences from pre-mRNA molecules and ligation of the internal ends of the molecules—splicing. Since the genomes (DNA) of higher organisms contain, alongside genes translated into corresponding proteins (exons), a significant number of nucleotide sequences that do not carry genetic information (introns), the splicing process ensures the removal of precisely these introns (non-translated sequences) from the primary transcripts:

Fig. 20.17. Splicing of the egg albumin gene pre-mRNA (Halkerston I.D.K., 1988).

Mature mRNA—that is, mRNA that has undergone 5'-capping, 3'-polyadenylation, splicing, and methylation of individual nucleotides—passes from the nucleus into the Cytoplasm as ribonucleoprotein complexes capable of interacting with ribosomes during Translation.

* The term "palindrome" (from Greek meaning "running back") refers to a word or sentence that reads the same backward as forward (e.g., "Madam, I'm Adam"). In Molecular Genetics, palindromes are defined as polynucleotide sequences (DNA or RNA) containing inverted repeats of nucleotide sequences with second-order axial Symmetry.

In DNA molecules, such palindromes can form secondary structures such as "cruciforms" or "hairpins," which serve as recognition sites for specific enzymes (such as Restriction Endonucleases—Chapter 23) and transcription termination signals. Upon transcription of palindromic regions, RNA transcripts are produced with mutually complementary sequences prone to forming hairpins (Fig. 20.16).