Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Gene Expression
DNA Transcription

Hereditary information, encoded in the language of nucleotide sequences, is stored in DNA in most organisms (except for Introduction/7.html">RNA-containing Viruses). The sequence of nucleotide triplets in genes determines the sequence of Amino Acids in Polypeptides or of ribonucleotides in transfer and ribosomal RNA molecules. For the genetic program to be realized—meaning the synthesis of necessary Proteins and RNAs—the Gene Expression machinery must be involved. Gene expression is defined as the synthesis of messenger RNAs and proteins. In this process, mRNAs act as intermediaries between DNA and protein: Protein Synthesis always occurs on single-stranded mRNAs (with the participation of Ribosomes), whereas the mRNAs themselves are always synthesized on double-stranded DNAs. Both processes are template-driven and subject to regulation, which significantly impacts the level of cellular METABOLISM.

The expression of all genes begins with the Transcription of their nucleotide sequence. Transcription is The process of translating information encoded in the language of deoxyribonucleotide sequences in the sense strand of DNA into the language of ribonucleotide sequences in mRNA. In this process, a specific region of one of the two DNA strands (the antisense strand) is used as a template for RNA Synthesis via complementary base pairing.

The Enzymes that catalyze transcription are DNA-dependent RNA polymerases. Notably, prokaryotes—such as Escherichia coli Cells—contain only a single type of this enzyme, which synthesizes all Three types of RNA (mRNA, tRNA, rRNA). In contrast, eukaryotes possess three different DNA-dependent RNA polymerases, each responsible for transcribing genes that encode Different types of cellular RNA. The process of transcription, as well as its enzymatic machinery, has been best studied in prokaryotes. Bacterial RNA polymerases are complex proteins consisting of several different subunits. The most thoroughly studied enzyme is the E. coli RNA polymerase holoenzyme, which contains five distinct polypeptide subunits: two α chains, one β and one β' chain, a σ chain, and an ω chain. An alternative form of the enzyme, known as the core enzyme or minimum enzyme, lacks the σ subunit. The core enzyme catalyzes the majority of DNA-to-RNA transcription reactions; however, it cannot initiate RNA synthesis at the correct site because it is unable to recognize promoter sites. Precise binding and initiation at promoters occur only after The addition of the σ subunit to the core enzyme, forming the holoenzyme.

Like other template-directed processes, transcription comprises three stages: initiation, elongation, and termination.

Transcription initiation. This process requires a holoenzyme, a specific nucleotide sequence in the DNA (the promoter), and a set of nucleoside triphosphates. Transcription is initiated upon The formation of a stable complex between the holoenzyme and a specific sequence called the promoter, which is located at the beginning of all transcriptional units. A promoter is a segment of the DNA molecule, approximately 40 nucleotide pairs in length, located immediately upstream of the transcription initiation site. It contains two important and relatively conserved sequences. One of these consists of six or seven NUCLEOTIDES (most often TATAAT) and is located at a distance of about 10 nucleotides from the first transcribed nucleotide (+1); this signal is usually designated as the -10 sequence, or the Pribnow box, named after its discoverer. At this site, RNA polymerase binds to DNA. The second sequence is located approximately 35 nucleotides upstream of the initiation site and serves as the promoter recognition region for RNA polymerase (Fig. 3.1).

When RNA polymerase binds to the promoter, a local unwinding of the DNA double helix occurs, forming an open promoter complex. Within this complex, The nucleotide sequence of the DNA sense, or (+), strand—oriented in the 5' → 3' direction—is copied. mRNA synthesis always begins with either an A or G nucleotide. The second, antisense DNA strand serves as the template for RNA chain synthesis (Fig. 3.2).

Transcription is analogous to Replication in the sense that the order of ribonucleotide incorporation is determined by complementary base pairing (Fig. 3.2). After the Formation of the first few phosphodiester bonds (usually 5–10), the σ subunit dissociates from the initiation complex, and further transcription is carried out by the core enzyme.

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Fig. 3.1. Structure OF THE E. coli promoter (explained in the text)

Fig. 3.2. The template principle of transcription

Transcription elongation. The growing RNA chain remains bound to the enzyme and base-paired at its growing end with a region of the template strand. The rest of the synthesized chain is attached neither to the enzyme nor to the DNA. As transcription continues, the core enzyme moving along the DNA strand acts like a zipper, unwinding The Double Helix, which then rewinds behind the enzyme to restore its original duplex structure. The DNA region "unmasked" by the enzyme spans only a few nucleotide pairs (Fig. 3.3).

RNA elongation proceeds in the 5'-to-3' direction along the template (-) strand, which is oriented in the 3'-to-5' direction (i.e., antiparallel). Transcription continues uninterrupted until the enzyme reaches a transcription termination site.

Transcription termination. DNA sequences that signal the cessation of transcription are called transcription terminators. They contain inverted repeats, causing the 3' ends of RNA transcripts to fold into hairpins of varying lengths (Fig. 3.4).

Fig. 3.3. DNA Transcription: the core enzyme operates within a local region of "unwound" DNA, synthesizing an mRNA chain in the 5'P → 3'OH direction

Fig. 3.4. Example of a hairpin within an mRNA molecule where transcription terminates. Complementary nucleotides are present within the stem of the hairpin

Two Types of termination signals have been discovered: ρ-dependent and ρ-independent terminators. ρ is an oligomeric protein that tightly binds to RNA and, in this state, hydrolyzes ATP to ADP and inorganic phosphate. In one model, the action of the ρ protein is explained by its binding to the synthesized RNA chain and moving along it in the 5' → 3' direction toward the site of RNA synthesis; the energy required for its movement is released during ATP Hydrolysis. If the ρ protein encounters a hairpin forming in the RNA, it halts the polymerase, which would otherwise continue transcription. The Mechanism of ρ-independent termination is less well understood and remains largely unclear.

In most cases, primary transcripts generated in the manner described above are not mature RNA molecules and require a maturation process known as RNA Processing. Processing differs significantly between prokaryotic and eukaryotic RNAs.

In prokaryotes, primary transcripts formed from the transcription of protein-coding genes function as mRNA without subsequent modification or processing. Moreover, mRNA Translation often begins even before the Synthesis of the 3' end of the transcript is complete. A completely different situation is observed for prokaryotic rRNA and tRNA molecules. In this case, clusters of rRNA or tRNA genes are frequently transcribed to form a single RNA chain. Specific Cleavage of the primary RNA transcripts and subsequent modification must occur to form mature, functional forms. These molecular events are referred to as RNA processing or post-transcriptional modification. The initial cleavage of primary transcripts into fragments containing either tRNA or 16S, 23S, or 5S rRNA sequences is carried out by the endonuclease RNase III. Its targets are short RNA duplexes formed by intramolecular base pairing in sequences flanking each RNA segment. These complementary sequences form hairpins at which RNase introduces cleavages, after which excess spacer region sequences are removed by other RNases. tRNA molecules are initially synthesized as pre-tRNAs, which are ~ 20% longer than mature tRNAs. Excess sequences located at the 5' and 3' ends are removed by ribonucleases Q and P. In addition, for the formation of a mature, functional tRNA, specific base modifications and the addition of one, two, or all three nucleotides of the 3'-CCA end (the acceptor stem) apparently must occur.

RNA maturation in eukaryotes is considerably more complex. First, eukaryotes possess a Nucleus separated from the Cytoplasm by a nuclear membrane. The Formation of primary transcripts takes place within The Nucleus; these transcripts are longer than the cytoplasmic mRNA participating in translation. Consequently, the formation of mature mRNA in eukaryotes must be preceded by the removal of introns from the hnRNA transcript sequence (a process known as splicing, from the English "to splice"). Following the removal of sequences corresponding to introns, the segments transcribed from exons are joined together. Splicing is catalyzed by protein-RNA complexes (snRNPs) that interact with hnRNA to form the spliceosome. It is believed that the RNA component possesses catalytic activity within the spliceosome. Such RNAs are called ribozymes. The splice site is determined within spliceosomes with high precision, since an error of even a single nucleotide can lead to structural distortion of the protein. For precise recognition, specific sequences—signals—are present within the introns.

In addition to splicing, eukaryotic mRNA undergoes modification: a "cap" is synthesized at the 5' end, which is a methylated guanosine triphosphate residue structure that protects the mRNA from degradation by 5'-exonucleases. At the 3' end of the pre-mRNA, a polyadenylate sequence 150–200 nucleotides long, known as the "tail," is synthesized. These structures take part in The regulation of EUKARYOTIC GENE EXPRESSION. The processing of rRNA and tRNA in eukaryotes is carried out similarly to that in prokaryotes.