C. Posttranscriptional processing
A series of modifications—capping, splicing, and polyadenylation—are required to stabilize the pre-mRNA, promote its nuclear export, and allow efficient translation. Mutations that change any of these processes are directly linked to genetic disease.
A 7-methylguanosine is linked to the first transcribed nucleotide through a 5'-5' triphosphate bridge to form a 5' methyl cap. After this occurs by a capping enzyme and an RNA (guanine-7-) methyltransferase, the mRNA cannot be degraded by exoribonucleases. Included in the regulation of capping are transcription factors that promote cap formation. Capping occurs at the initiation of transcription. Following the export of mRNA to the cytosol, the 5' methyl cap binds to a translation factor (eIF4E), which is the first step of mRNA recruitment to the 40S ribosome subunit.
The primary transcript of most genes includes exons and introns. Conserved sites at each end of introns are recognized for removal of the introns by a process known as splicing. This occurs in the nucleus prior to the transport of mature mRNA to the cytosol. One of the several types of RNAs localized in the nucleus of the cell is small nuclear RNA (snRNA). Five specific snRNAs are essential for binding to more than 100 non-small nuclear ribonuclear protein-splicing factors to form small nuclear ribonucleoproteins (snRNPs). Assembling snRNPs involves transcription of snRNAs that are then capped with trimethylguanosine—a different cap than other mRNAs. The transcript is transported to the cytosol where it associates with several uridine-rich proteins (U1, U2, U4, U5, and U6), and the cap is modified to reenter the nucleus. snRNPs are called U1–U5 snRNPs to designate the protein association.
A spliceosome is composed of a complex of snRNPs and accessory protein factors and is the catalytic unit that creates splicing. The binding of the snRNPs and factors at specific sequences causes the intron to form a lariat to bring the ends into closer proximity. The exact positioning allows two consecutive transesterification reactions to occur followed by ligation. The ends of two exons align properly for recognition of each three nucleotides (codon) destined to become an amino acid during translation. The spliced intron is degraded and the nucleotides recycled.
An extra level of complexity is added to splicing by alternative splicing in which an mRNA can be spliced differently, thereby leading to a different assortment of exons; each different assortment of exons is a different mRNA (Figure 1.17). Different regulatory sequences, known as splicing enhancers or silencers, designate whether an exon is included under certain conditions. Serine–arginine-rich proteins bind to these sequences to regulate alternative splicing. In other cases, cell signaling can direct alternative splicing.
Alternative splicing provides different transcripts from one primary transcript.
Modified from Figure 19.3, Harvey RA, Ferrier DR. LIR Biochemistry, 5th ed. Baltimore, MD: Lippincott Williams & Wilkins, 2011
Transcription termination for all coding genes, with the exception of some histones, occurs following the synthesis of a poly(A) tail at the 3' end of the transcript. Polyadenylation is a two-step process that includes endonucleolytic cleavage of the transcript and subsequent posttranscriptional poly(A) addition. Changes in the length of the poly(A) tail, which is normally about 250 nucleotides, can affect the stability of the mRNA and its translational capacity; shortened tails are associated with mRNA instability and increased turnover.
Transcription
Transcription is the process in which DNA information is transferred to pre-mRNAs by RNA polymerases.
1. Preinitiation complex
RNA polymerase II is composed of 12 subunits and is the target of regulation by multiple transcription factors that specify which genes are transcribed. The promoter is the binding site for transcription factors that form a preinitiation complex (PIC). RNA polymerase II does not bind directly to the promoter sequence but to the PIC. Enhancer sequences can, when bound, also modify the rate of initiation complex formation. The rate of transcription is controlled by the stability of the complex, which can dissociate easily from the promoter.
Acetylation of histones in the nucleosome allows transcription to occur through unwinding of the helix. Some genes, such as constitutive genes, are transcribed over a significant amount of time. For others, expression occurs in transcriptional bursts represented as fluctuations between active and inactive transcription. Elongation occurs as the RNA polymerase reads the DNA template from the 5' to 3' direction and forms a complementary RNA strand. The new strand is distinguished from the template strand by the presence of uracil (U) for thymine bases pairing with adenine (.
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DNA is used as a template to create a messenger RNA (mRNA) molecule.
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This occurs in the nucleus.
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An enzyme called RNA polymerase binds to a specific region of DNA called the promoter, located at the beginning of a gene.
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Transcription factors help RNA polymerase recognize the promoter and begin transcription.
The initiation site of a gene is the specific location where transcription begins—in other words, where RNA polymerase starts synthesizing RNA based on the DNA template. This site is also known as the:
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Transcription start site (TSS)
Key points about the initiation site:
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It is usually designated as +1 on the DNA sequence.
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It is located downstream of the promoter region, which is the sequence RNA polymerase and transcription factors bind to in order to initiate transcription.
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The initiation site marks the first nucleotide that is transcribed into RNA.
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In eukaryotes, it's often located just after the TATA box (a common promoter sequence), although the exact position varies between genes.
In summary:
The initiation site (or transcription start site) is the first base of the gene that gets transcribed into RNA, and it plays a central role in gene expression.
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The initiation site of a gene is the specific location where transcription begins—in other words, where RNA polymerase starts synthesizing RNA based on the DNA template. This site is also known as the:
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Transcription start site (TSS)
Key points about the initiation site:
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It is usually designated as +1 on the DNA sequence.
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It is located downstream of the promoter region, which is the sequence RNA polymerase and transcription factors bind to in order to initiate transcription.
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The initiation site marks the first nucleotide that is transcribed into RNA.
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In eukaryotes, it's often located just after the TATA box (a common promoter sequence), although the exact position varies between genes.
In summary:
The initiation site (or transcription start site) is the first base of the gene that gets transcribed into RNA, and it plays a central role in gene expression.
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2. Elongation:
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RNA polymerase moves along the DNA template strand, unwinding the DNA and synthesizing a single-stranded RNA molecule.
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It adds RNA nucleotides that are complementary to the DNA template (A pairs with U, C with G, etc.).
- Termination:
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RNA polymerase continues until it reaches a termination signal in the DNA.
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The RNA strand is released and is now called messenger RNA (mRNA) (in the case of protein-coding genes).
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- RNA Splicing
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mRNA splicing is the process by which non-coding regions (introns) are removed from a pre-mRNA transcript, and the coding regions (exons) are joined together to form a mature messenger RNA (mRNA) molecule. This happens in eukaryotic cells before the mRNA is exported from the nucleus for translation.
What mRNA Splicing Accomplishes:
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Removes Introns:
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Introns are non-functional sequences that do not code for proteins. Splicing removes them so the mRNA can be correctly translated.
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Creates a Continuous Coding Sequence:
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Exons are stitched together to form a proper, continuous coding sequence for the ribosome to read.
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Enables Alternative Splicing:
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The same gene can be spliced in different ways to produce multiple proteins (isoforms) from a single gene. This greatly increases protein diversity.
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Prepares mRNA for Export and Translation:
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Splicing is part of the overall maturation of mRNA, which includes adding a 5′ cap and a poly-A tail. Only mature mRNA is exported to the cytoplasm for translation.
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In short, mRNA splicing ensures the genetic message is accurate and versatile, and it's essential for proper gene expression in eukaryotic organisms.
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The initial RNA transcript (pre-mRNA) is spliced (introns removed, exons joined).
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A 5' cap and a poly-A tail addition at the 3' end to protect the mRNA and aid in translation.
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Splicing to remove non-coding sequences (introns) and join coding sequences (exons)
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Key Steps in Gene Transcription:
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Initiation:
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An enzyme called RNA polymerase binds to a specific region of DNA called the promoter, located at the beginning of a gene.
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Transcription factors help RNA polymerase recognize the promoter and begin transcription.
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- Elongation:
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RNA polymerase moves along the DNA template strand, unwinding the DNA and synthesizing a single-stranded RNA molecule.
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It adds RNA nucleotides that are complementary to the DNA template (A pairs with U, C with G, etc.).
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Result:
The mature mRNA exits the nucleus and travels to the ribosome, where it guides translation — the next step in protein synthesis.