Fundamentals of Biochemistry - A. A. Anisimov 1986

Nucleic Acids
RNA Synthesis

4.8.1. METABOLISM/31.html">Transcription. Prokaryotic Transcription. Transcription is the process by которой Genetic information enclosed in DNA is «rewritten» into single-stranded RNA molecules, which are subsequently transported to Ribosomes.

Units of transcription contain information regarding The Structure of one or more Proteins. A DNA region containing the structural information for a single protein is called a Cistron, or structural Gene. Transcription is regulated by special regulatory sequences within the DNA. The regulatory zone includes a promoter, an operator, and frequently other control elements (Fig. 4.17).

The promoter contains the site for the initial, stable binding of DNA-dependent RNA polymerase to DNA. The operator is a regulatory region that binds repressors—proteins that control mRNA synthesis in accordance with cellular demands. The operator and promoter sometimes partially overlap. In certain transcription units (operons), a so-called leader region is located between the operator and the structural genes. It is transcribed but, as a rule, not translated. This region houses the ribosome-binding site on the mRNA and an attenuator, which regulates transcription by influencing the interaction between RNA polymerase and the DNA template. A terminator is located downstream of the structural genes.

A DNA nucleotide sequence flanked by a promoter and a terminator, encoding a single mRNA molecule and controlled by an operator, is referred to as an Operon. Prokaryotes are known to possess operons containing several cistrons (genes) that encode the Enzymes of a single metabolic pathway. Due to the presence of the regulatory zone, all cistrons are switched on and off simultaneously. The operon concept was formulated in 1959 by F. Jacob and J. Monod and remains fundamental to understanding the mechanisms of gene regulation in prokaryotes. The Genome also contains transcription units that operate constitutively without being controlled by any operator.

DNA-dependent RNA Synthesis can be divided into several stages that collectively constitute the transcription cycle, which has been studied in detail in prokaryotic systems. The First stage of transcription, initiation, involves the interaction of RNA polymerase with template DNA. RNA polymerase can bind to any region of DNA, forming a non-specific, weak complex with a short half-life. Through a series of association-dissociation events at random DNA sequences, the RNA polymerase locates the promoter. Within the promoter region, a closed, stable complex between DNA and RNA polymerase is initially formed. This is followed by local Denaturation of the DNA, granting RNA polymerase direct access to the DNA bases and resulting in an open complex.

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Fig. 4.17. Structure of a prokaryotic operon:

P — promoter, O — operator, A, B, C — cistrons, t — terminator, R — regulatory gene

Because mutually complementary strands of a DNA molecule are antiparallel and the code lacks complementary degeneracy, both DNA strands cannot encode the same protein. It is generally accepted that either only one of the two strands is transcribed, or both are transcribed, but only one of the resulting RNA molecules serves as a template, while the second performs alternative Functions. The DNA strand complementary to the mRNA is usually called the coding strand, and the second strand is termed the template (non-coding) strand. Since transcription is asymmetric (proceeding on only one of the two strands), it can be inferred that the promoter is also fixed on only One DNA strand, possesses an asymmetric structure, and determines the direction of RNA polymerase movement—i.e., its choice of the coding strand.

Upon the availability of appropriate ribonucleoside triphosphates, RNA synthesis commences. The first NUCLEOTIDES in Transcription initiation are almost always A or G. A ternary complex consisting of DNA, RNA polymerase, and the growing RNA chain is formed. The promoter is not transcribed, whereas the operator is partially transcribed in some instances.

Elongation (chain growth) of RNA proceeds in the direction. Ribonucleotides are attached sequentially to the 3'-OH end, one by one, According to the DNA template. The rate of elongation in E. coli Cells in vivo is 45–50 nucleotides per second at 37°C.

RNA synthesis termination is triggered by a specific DNA nucleotide sequence known as a terminator or stop signal. The structure of terminators has not been fully elucidated, though it is known to include extended blocks. In E. coli, stop signals of another type have been discovered that function only in the presence of a protein designated as the p-factor. This protein has an M = 50,000 and is typically not bound to RNA polymerase.

With its participation, the RNA chain is released from the DNA template, although the enzyme remains bound to DNA. Additional factors are required for the complete release of the enzyme. Another termination-involved protein, the kappa particle, has also been isolated.

In bacterial cells, ribosomes attach to the nascent mRNA transcript as it begins to detach from the template. They facilitate the dissociation of mRNA from the template and initiate Protein Synthesis. This forms a unified transcription-Translation complex, which has been successfully visualized using Electron Cell/15.html">Microscopy (Fig. 4.18).

Fig. 4.18. Transcription and translation in E. coli cells (based on electron microscopy data).

mRNA chains are shown growing along the double-stranded DNA. At the top, where transcription has just begun, the mRNA chains are shorter, whereas further down, where transcription has proceeded for a longer duration, they are longer. Ribosomes attach to the mRNA chains, and Polypeptide chain synthesis begins

Features of Eukaryotic Transcription. While transcription and translation are coupled in prokaryotes, these processes are separated in time and space in eukaryotes. The Cell Nucleus plays a crucial role here by compartmentalizing the transcription and translation machinery. When describing transcription in eukaryotes, the term «operon» can only be used quite loosely, because genes determining the Structure of enzymes in a single metabolic pathway are not necessarily adjacent and may even be localized on different Chromosomes. Consequently, Mutations interrupting the same metabolic pathway in eukaryotes are also «scattered» throughout the genome. Eukaryotic structural genes feature large series of regulatory regions rather than single ones. Furthermore, the enzyme systems that read genomic information in pro- and eukaryotes differ, as do the Processing steps converting primary transcription products into mature mRNA molecules.

Most cytoplasmic RNA molecules are initially synthesized in The Nucleus as high-molecular-weight precursors—pre-mRNA and pre-rRNA. These precursors are significantly longer than cytoplasmic RNAs and undergo processing (maturation). Transcription in Eukaryotic organisms follows roughly the same stages as in prokaryotes, but it is carried out by three distinct RNA polymerases. Transcription by RNA polymerases I and III is less thoroughly studied; however, it has been established that transcription initiation at various genes is determined by internal regions that share structural similarities yet lie at varying distances from the transcription start site.

Eukaryotic RNA polymerase II catalyzes the synthesis of all pre-mRNAs. The functional mechanism of RNA polymerase II largely mirrors prokaryotic systems. The analysis of transcription products has revealed characteristic structural features in both the structural and regulatory Regions of the genome. Histone genes have been studied in the greatest detail, followed to a somewhat lesser extent by globin, Ovalbumin, and conalbumin genes.

Most genes in this group share a conserved sequence (TATAAA or the shorter TATA or ATA), known as the Goldberg-Hogness (TATA) box. It is located 21–28 nucleotide pairs upstream of the transcription start site. GC-rich regions flank both sides of the AT-rich sequence. The TATA box is presumed crucial for transcription initiation, thus functioning as a promoter. It determines the Selection of the transcription start site without affecting its efficiency, which is why it is sometimes called a selector. At a specific distance downstream of the TATA sequence lies the initiator region, where mRNA transcription directly begins, concluding prior to the ATG codon. Approximately 100–300 nucleotide pairs upstream/downstream from the start site, beyond the selector, lies another functionally vital region—the modulator (there may be multiple modulators). It determines the synthesis rate of structurally invariant mRNA molecules exclusively in the presence of the selector and initiator (Fig. 4.19).

Transcription initiation can also be influenced by more distant genomic regions. The true promoter of genes transcribed by RNA polymerase II is apparently assembled from multiple genomic regions through a specific Chromatin conformation. Numerous cellular protein factors, which remain poorly understood, are likewise responsible for the precision and efficiency of transcription.

Fig. 4.19. Structure of a eukaryotic transcription unit:

A and B — modulators, B — selector, C — initiator, t — transcription terminator, poly(A) tract, which presumably acts as a primer for polyadenylation during processing; 5' — first transcribed codon; AT — codon initiating translation on the ribosome; TC — Translation termination codon; dark regions denote exons, light regions denote introns

The structural zones of eukaryotic genes also possess certain features that distinguish them from bacterial structural genes. Within the regions encoding protein structures, there are gaps known as introns. These alternate with exons, which are gene segments encoding the sequences of mature mRNAs (Fig. 4.19). The first exon, if it does not encode a polypeptide but instead serves as a template for the 5'-untranslated region of the mRNA, is referred to as the leader sequence. The number of exons varies widely among different genes. It has been hypothesized that, in A number of cases, exons correspond to specific domains within the encoded proteins. This has been demonstrated, for example, in studies of immunoglobulin genes. It is possible that the rearrangement of large blocks (exons) within genes represents one of the pathways of their evolution.

Transcription termination in Eukaryotic cells is not yet fully understood. Some insights have been gained, for example, from the analysis of histone genes. In the region adjacent to the 3'-end of the structural gene zone, at a distance of 23–47 Base Pairs from its terminal codon, a conserved region has been discovered consisting of two short palindromes separated by a TTTT sequence. It is suggested that the RNA forms a hairpin loop in this region, which is involved in termination.

The work of American cytologist O. Miller (1969) made it possible to visualize units of the transcription process using electron micrographs. The scientist isolated active Genes encoding ribosomal RNA from amphibian oocytes. A diagram based on this microphotograph depicts DNA strands with RNA polymerase molecules attached to them and RNA strands dangling from the polymerase molecules (Fig. 4.20). The diagram demonstrates that transcription is carried out simultaneously by 80–100 RNA polymerase molecules that bind sequentially to the promoter.

Fig. 4.20. Formation of RNA molecules on a DNA template corresponding to a single gene

4.8.2. mRNA Processing. In eukaryotic cells, primary transcripts are converted into mRNA through processing. Pre-mRNAs constitute the main fraction of heterogeneous nuclear RNA (hnRNA), which was first isolated in 1961 by G. P. Georgiev and coworkers. Pre-mRNAs contain from 5,000 to 50,000 nucleotides, whereas mRNAs are relatively short, with an average size of about 2,000 nucleotides. Each mRNA encodes a single polypeptide chain, meaning it is monocistronic. Typically, each pre-mRNA molecule gives rise to only a single mRNA molecule, while the majority of the pre-mRNA chain (sometimes up to 90% or more), corresponding to the non-coding region of the DNA, undergoes enzymatic Cleavage into free nucleotides and does not enter the Cytoplasm. These modifications constitute a part of processing.

Processing (post-transcriptional modification) includes: 1) the removal of excess terminal sequences, 2) the cleavage of long primary transcripts and the "excision" of segments transcribed from introns, 3) The addition of nucleotides to the 3'-end of the transcript, 4) the addition of nucleotides to the 5'-end of the transcript, and 5) the modification of bases within the transcript.

Processing takes place in the nucleus. A sequence of 150–200 adenylyl nucleotides—the poly(A) tail—is added to the 3'-end of the pre-mRNA (with the participation of a specialized enzyme), while a cap is added to the 5'-end (see Section 4.4.3). The excision of introns from the primary transcript is accompanied by splicing. The Mechanism of splicing has not been fully elucidated. It is hypothesized to involve a specific U1 RNA and a certain enzyme. [U1 RNA recognizes the junction sites of introns and exons, interacting with them through complementary base pairing to form a pre-mRNA—U1 RNA–enzyme complex. This brings the two regions of the pre-mRNA transcribed from neighboring exons into close proximity.

The region transcribed from the intron forms a loop (Fig. 4.21). The resulting hybrids can serve as targets for RNase attacks specific to double-stranded RNA regions. Following the excision of the redundant region (i.e., the one transcribed from the intron), specialized ligases join the two ends of the cleaved pre-mRNA molecule.

As a result of processing, the pre-mRNA yields an mRNA molecule featuring a 5'-cap, a universal 3'-poly(A) block, and a single coding sequence. Processing is not characteristic of Prokaryotic Cells.

Splicing occurs in eukaryotic cells during The Biosynthesis of mRNA, tRNA, and rRNA alike. For example, certain Yeast tRNAs undergo splicing, even though their introns are very small in size. In Drosophila, rRNA genes also contain an intron.

4.8.3. Ribonucleoprotein Complexes (RNP Complexes). Immediately after transcription, RNA molecules bind with proteins to form compact structures. RNP complexes containing hnRNA are called nuclear informosomes (hnRNPs). The protein-to-RNA ratio in these complexes is 4:1.

In addition to hnRNA, low-molecular-weight stable RNAs with sedimentation coefficients ranging from 4.5S to 6.5S have been found within hnRNPs. They are closely associated with both the hnRNA molecules themselves and the Proteins of the RNP particles.

The general Structural Organization of hnRNPs can be represented as follows. Numerous monoparticles, arranged in a non-strictly regular manner, form along the hnRNA chain. These 30–40S particles consist of a protein globule (informopher) around The surface of which a small segment (600 nucleotides) of the hnRNA chain is "wound" in a specific way. Monoparticles alternate with heterogeneous (30–200S) RNP complexes. Under electron microscopy, such structures appear as fibrils extending from chromatin with numerous repeating RNP particles.

Fig. 4.21. Putative structure formed during splicing.

Dashed lines indicate Hydrogen Bonds formed between complementary nitrogenous bases. Dark regions represent pre-mRNA sequences transcribed from exons, and light regions represent those transcribed from introns.

¹ The word "splicing" is borrowed from English nautical terminology, where it refers to joining ropes without tying a knot.

It is hypothesized that specific Tertiary and Quaternary structures of hnRNA, driven by Protein Interactions, facilitate the precise progression of processing and splicing. It has been established that hnRNA processing takes place within RNP particles. mRNPs appear in the cytoplasm following the processing of hnRNPs. Free mRNP particles (cytoplasmic informosomes) or mRNPs associated with Polysomes (polysomal informosomes) are found here. In these structures, the mass ratio of protein to RNA ranges from 3:1 to 4:1. mRNP particles contain mRNA molecules of various sizes. Polysome-associated mRNAs are actively translated. Polysomal and free cytoplasmic informosomes contain no more than 15–20 different proteins.

Two main proteins are found in polysomal informosomes. Free informosomes contain a larger set of proteins, among which are translation-inhibiting proteins. mRNP proteins differ significantly from hnRNP proteins. The transition from the nucleus to the cytoplasm is accompanied by a shift in the protein component of the RNP particles. Proteins that bind to informosomes mask the mRNA and ensure its storage in the cytoplasm in a untranslated state. The transition of mRNPs from informosomes to polysomes must also be accompanied by changes in protein composition: the dissociation or modification of repressor proteins and the binding of activator proteins (initiation factors, etc.).

A. S. Spirin (1966) suggested that informosomes serve as a storage form for mRNA in the cytoplasm prior to its entry into polysomes and subsequent translation. In this form, for instance, the mRNA of an egg cell is stored in an inactive state until Fertilization, and in seeds until germination.

Thus, in eukaryotic cells, mRNA is always found in a complex with proteins. The RNP complex is the sole form of mRNA existence in animal and plant cells, from the moment of pre-mRNA synthesis in the nucleus to the degradation of mRNA in the cytoplasm.

4.8.4. Synthesis of Ribosomal and Transport RNAs. All ribosomal and Transfer RNAs are synthesized on a DNA template. In *E. coli*, Ribosomal RNAs are produced as a large 30S precursor. This gives rise to all rRNAs—16S, 23S, and 5S. During maturation, rRNAs undergo intensive methylation. Transfer RNAs in prokaryotes are also transcribed as precursors containing one or more tRNAs. For example, T4 phage genes encode a precursor for two tRNAs simultaneously—Proline and Serine. It is "cleaved" into monomers, from which excess segments are subsequently removed. If the cleavage products lack the CCA sequence, it is synthesized by a dedicated enzyme.

In eukaryotic cells, rRNAs are encoded by a chromosomal region that forms the nucleolus—the nucleolar organizer. Here, the region encoding the 28S, 18S, and 5.8S rRNAs is repeated several hundred times. RNA polymerase I generates a large precursor from the transcribed region. Several hundred nucleotides located at the 5'-end are extremely rapidly removed from it. The remaining precursor undergoes processing. In mammalian cells, 8 pre-rRNAs of varying sizes (ranging from 45S to 12S) have been detected, leading to the hypothesis that at least three processing pathways exist. The cleavage sites of the precursor (45S RNA) are identical in all cases; There are five cleavage sites in total, but the order of cleavage may vary. Typically, the 45S RNA is cleaved into two parts by an endonuclease. Subsequently, Other Enzymes remove the spacers (intervening sequences). One half yields the 18S rRNA, while the other yields the 28S and 5.8S rRNAs held together by hydrogen bonds.

The synthesis of 5S rRNA and tRNA is catalyzed by RNA polymerase III. Pre-5S rRNA contains a small number of extra nucleotides at the 3'-end of the molecule, which are removed during processing. tRNA precursors contain redundant sequences that can be located at both ends of the molecule as well as internally. During pre-tRNA Processing, these extra sequences are cleaved off, base modifications take place, the intervening sequence (if present) is excised, and splicing occurs. The CCA sequence is added to the 3'-end of the pre-tRNA. All these processes take place in the nucleus.