Chapter 13: The Genetic Code and Transcription PDF

Chapter 13 The Genetic Code and Transcription Genetic information is stored in DNA and encoded in a nearly universal form across all living organisms. The genetic code is transferred from DNA to RNA through transcription, where it exists as triplet codons, each composed of three ri...

Chapter 13 The Genetic Code and Transcription Genetic information is stored in DNA and encoded in a nearly universal form across all living organisms. The genetic code is transferred from DNA to RNA through transcription, where it exists as triplet codons, each composed of three ribonucleotides. RNA's four ribonucleotides can form 64 different three-letter sequences, most of which encode one of the 20 amino acids in proteins. Some codons serve as signals to start or stop protein synthesis. Transcription in bacteria is simpler than in eukaryotes, where the initial RNA transcript requires processing before translation. Learning Objectives The genetic code is composed of triplet codons, each specifying a particular amino acid. Experimental evidence, such as the use of synthetic mRNAs in cell-free systems, supports the triplet nature of the genetic code. Adding or deleting nucleotides can shift the reading frame, altering the downstream amino acid sequence. Crick’s wobble hypothesis explains how some tRNA molecules can recognize multiple codons due to flexible base pairing at the third codon position. Single base changes in DNA can lead to specific amino acid substitutions in proteins. Mutations can be identified by comparing wild-type and mutant mRNA or amino acid sequences. The mRNA codon and tRNA anticodon are complementary; knowing one allows you to determine the other. There are exceptions to the universal genetic code in certain species, where specific codons have different meanings. The central dogma of molecular genetics describes the flow of genetic information from DNA to RNA to protein. Transcription involves specific DNA regions and enzymes for initiation, elongation, and termination in bacteria. RNA molecules can be traced back to their DNA templates, and their sequences can predict the resulting amino acids. Eukaryotic transcription differs from bacterial transcription in promoter sequences and additional processing steps. Eukaryotic post-transcriptional processing includes 5’ capping, 3’ polyadenylation, and intron splicing, which affect mRNA stability. mRNA, tRNA, and rRNA undergo different processing steps. RNA splicing removes introns, while RNA editing alters nucleotide sequences. Electron microscope studies provided visual evidence of transcription processes. 13.1 The Genetic Code Uses Ribonucleotide Bases as “Letters” The genetic code is linear, using ribonucleotide bases in mRNA derived from complementary DNA bases. Each mRNA "word" consists of three ribonucleotide letters, forming a triplet code. Each triplet codon specifies one amino acid, making the code nearly unambiguous. The code is degenerate; most amino acids are specified by more than one triplet codon. There are specific "start" and "stop" codons that initiate and terminate translation. The code is commaless, meaning codons are read sequentially without internal punctuation. The code is nonoverlapping; each ribonucleotide is part of only one triplet.  The code is colinear; the sequence of codons in mRNA determines the sequence of amino acids in the protein. The code is nearly universal, used by almost all forms of life with minor exceptions. 13.2 Early Studies Established the Basic Operational Patterns of the Code In the late 1950s, researchers initially thought DNA might directly encode proteins during synthesis. Ribosomes were identified, leading to the hypothesis that DNA information was transferred to ribosomal RNA (rRNA) for protein synthesis. This hypothesis was disproven as evidence showed the need for an unstable intermediate template, unlike the stable rRNA. In 1961, François Jacob and Jacques Monod proposed the existence of messenger RNA (mRNA). mRNA was discovered to be the intermediate that carries genetic information from DNA to proteins. The genetic code in mRNA, composed of four ribonucleotides, specifies the 20 amino acids used in protein synthesis. The Triplet Nature of the Code Sydney Brenner theorized that the genetic code must be triplet-based because three-letter words are the minimal use of four nucleotides to specify 20 amino acids. A code of four nucleotides taken two at a time provides only 16 unique code words, which is insufficient for 20 amino acids. A triplet code yields 64 unique words which is more than enough for the 20 amino acids and simpler than a four-letter code, which specifies 256 words. Experimental evidence by Francis Crick and colleagues using phage T4 supported the triplet nature of the code. Frameshift mutations, caused by the addition or deletion of nucleotides, shift the reading frame during translation. Crick's research showed that the gain or loss of one or two nucleotides caused a frameshift mutation, but the reading frame was reestablished when three nucleotides were involved, confirming the triplet code. 13.4 The Coding Dictionary Reveals Several Interesting Patterns among the 64 Codons The genetic code consists of 61 triplet codons that correspond to amino acids. There are three additional codons that serve as termination signals and do not specify any amino acid. The genetic code is degenerate, meaning most amino acids are specified by multiple codons. Serine, arginine, and leucine are each encoded by six different codons, while tryptophan and methionine are encoded by single codons. Codons specifying the same amino acid often share the first two nucleotides, differing only in the third position. Example: Phenylalanine is specified by UUU and UUC differing only in the third nucleotide. Example: Valine is specified by GUU, GUC, GUA, and GUG, differing only in the third nucleotide. Crick's wobble hypothesis suggests that the first two nucleotides of a codon are more critical for tRNA binding than the third. The hypothesis proposes that the third position of the codon allows for more flexible base-pairing rules, leading to less strict adherence to base-pairing rules. Anticodon–Codon Base-Pairing Rules The wobble hypothesis explains that the relaxed base-pairing requirement allows a single tRNA anticodon to pair with multiple mRNA codons. At the first position (5' end) of the tRNA anticodon, the base U can pair with A or G at the third position (3' end) of the mRNA codon. Similarly, G at the first position of the tRNA anticodon can pair with C or U at the third position of the mRNA codon. Inosine (I), a modified base in tRNA, can pair with A, U, or C at the third position of the mRNA codon. Due to these wobble rules, only about 30 different tRNA species are needed to accommodate the 61 codons that specify amino acids. In bacteria, there are 30 to 40 tRNA species, while in eukaryotes, there are 41 to 55 tRNA species. The Ordered Nature of the Code The ordered genetic code refers to the pattern where chemically similar amino acids share one or two middle bases in their codon sequences. Hydrophobic amino acids like valine and alanine often have U or C in the second position of their triplets. Lysine, a positively charged amino acid, is specified by the codons AAA and AAG. Changing the middle base from A to G (resulting in AGA and AGG) specifies arginine, another positively charged amino acid. This ordered pattern helps buffer the effects of mutations on protein function, as changes in the second base often result in amino acids with similar chemical properties, minimizing functional disruption. Punctuating the Code: Initiation and Termination Codons Initiation of protein synthesis in vivo is highly specific, starting with the amino acid methionine. Methionine is inserted into all polypeptide chains, formylated in bacteria and unformylated in eukaryotes. The codon AUG is the primary initiator (start) codon for methionine. Rarely, GUG and UUG codons can also specify methionine during initiation, though GUG usually encodes valine and UUG encodes leucine. Three codons (UAG, UAA, and UGA) serve as termination (stop) codons, signaling the end of translation. Stop codons do not code for any amino acid and are not recognized by tRNA molecules. Translation terminates when a stop codon is encountered, releasing the polypeptide from the ribosome. 13.6 The Genetic Code Is Nearly Universal Initially, it was believed that the genetic code was universal across all organisms, including viruses, bacteria, archaea, and eukaryotes. mRNA and translation machinery appeared similar across these organisms, as evidenced by bacterial cell- free systems translating eukaryotic mRNAs. Early recombinant DNA technology showed that eukaryotic genes could be transcribed and translated in bacterial cells. Eukaryotic mRNAs from mice and rabbits were successfully translated in amphibian eggs. The amino acid sequences of many eukaryotic proteins, such as hemoglobin, matched the coding dictionary derived from bacterial studies. In 1979, studies on mitochondrial DNA (mtDNA) from yeast and humans revealed exceptions to the universal genetic code. Sequencing of cloned mtDNA fragments showed that the codon UGA, typically a stop codon, codes for tryptophan in yeast and human mitochondria.  In human mitochondria, the codon AUA, which usually codes for isoleucine, directs the insertion of methionine. In yeast mitochondria, the codon CUA, which normally codes for leucine, results in the insertion of threonine. These codon changes often involve shifts in the recognition of the third, or wobble, position. The evolutionary trend suggests a reduction in the number of tRNAs needed in mitochondria, with human mitochondria encoding only 22 tRNA species. These exceptions highlight the diversity and adaptability of genetic coding across different species and cellular environments. 13.7 Different Initiation Points Create Overlapping Genes The genetic code is nonoverlapping, meaning each ribonucleotide is part of only one codon. A single mRNA can have multiple initiation points for translation, potentially creating different reading frames. Multiple reading frames within the same mRNA can specify more than one polypeptide, leading to overlapping genes. 13.8 Transcription Synthesizes RNA on a DNA Template Proteins are the end products of many genes, and understanding genetic expression involves studying how DNA specifies proteins. The process begins with transcription, where genetic information in DNA is transferred to RNA, resulting in an mRNA molecule complementary to the gene sequence. Each triplet codon in mRNA is complementary to the anticodon of its corresponding tRNA, which inserts the correct amino acid during translation. Transcription is crucial as it initiates the flow of genetic information within the cell. Key findings supporting RNA as an intermediate molecule include: - DNA is located in the nucleus, while protein synthesis occurs in the cytoplasm. - RNA is synthesized in the nucleus and is chemically similar to DNA. - mRNA migrates to the cytoplasm where translation occurs. These observations led to the understanding that genetic information in DNA is transferred to RNA, which then directs protein synthesis. 13.9 RNA Polymerase Directs RNA Synthesis RNA polymerase is the enzyme responsible for synthesizing RNA on a DNA template, discovered in 1959 by several researchers, including Samuel Weiss. RNA polymerase uses nucleoside triphosphates (NTPs) as substrates, which contain ribose instead of deoxyribose, unlike DNA polymerase. RNA synthesis does not require a primer to initiate, unlike DNA synthesis. The overall reaction for RNA synthesis can be summarized as n(NTP)→(NMP)n+n(PPi), where NTPs are polymerized into nucleoside monophosphates (NMPs) forming a polynucleotide chain. Nucleotides are linked by 5' to 3' phosphodiester bonds, and the energy for the reaction comes from cleaving the triphosphate precursor into the monophosphate form, producing inorganic diphosphates (PPi). The sequential addition of ribonucleotides during transcription is represented by (NMP)n+NTP→(NMP)n+1+(PPi) where each step adds one ribonucleotide to the growing chain. E. coli RNA polymerase consists of core enzyme subunits: two α, β, β', and ω. The holoenzyme includes an additional σ subunit, with a molecular weight of nearly 500 kDa. The β and β' subunits are crucial for the catalytic mechanism and active site of transcription, while the σ factor regulates the initiation of RNA transcription.  E. coli has a single form of the core enzyme but multiple σσ factors, leading to different polymerase holoenzymes. Eukaryotes have three distinct forms of RNA polymerase, each with more polypeptide subunits than bacterial RNA polymerase. The process of transcription in bacteria will be discussed, with eukaryotic transcription covered later. Promoters, Template Binding, and the σ Subunit Transcription synthesizes a single-stranded RNA molecule complementary to the DNA template strand. The DNA strand used for transcription is the template strand, while the complementary strand is the coding strand. The coding strand and the RNA molecule have the same 5' to 3' sequence, except RNA has uridine/uracil (U) instead of thymidine/thymine (T). In bacteria, transcription begins with template binding, where RNA polymerase's σ subunit recognizes promoter sequences upstream of the gene. Promoters are crucial for transcription efficiency and are located in the 5' region, upstream from the coding sequence. The RNA polymerase binds to about 60 nucleotide pairs, unwinding the DNA helix locally. The transcription start site is indicated as position +1. Promoter sequences include consensus sequences, which are conserved DNA sequences critical for biological processes. In bacteria, two key consensus sequences are: - TATAAT (Pribnow box) at the −10 region. - TTGACA at the −35 region. Mutations in these regions can severely diminish transcription. Cis-acting elements are regulatory sequences on the same DNA molecule as the gene they regulate, while trans-acting factors are proteins that bind to these elements to influence gene expression. RNA polymerase binding to promoters varies, leading to different levels of gene expression. This variation is due to sequence differences in promoters, resulting in strong or weak promoters. The σ subunit in bacteria, such as σ70 in E. coli, recognizes most gene promoters. Other σ factors (e.g., σ32, σ54, σS, σE) regulate gene expression under specific conditions like heat or starvation, each recognizing different promoter sequences. Initiation, Elongation, and Termination of RNA Synthesis in Bacteria RNA polymerase binds to the promoter and converts DNA from a double-stranded form to an open structure, exposing the template strand. RNA synthesis begins with the insertion of the first ribonucleoside triphosphate at the start site, forming the 5' end of the transcript. RNA polymerization proceeds in the 5' to 3' direction, linking ribonucleotides via phosphodiester bonds. An 8-bp DNA/RNA duplex forms temporarily, with chains running antiparallel. The σ subunit dissociates after initial ribonucleotides are added, and elongation continues under the core enzyme. In E. coli, elongation occurs at about 50 nucleotides/second at 37°C. RNA polymerase performs proofreading, recognizing and correcting mismatches by backing up and removing noncomplementary bases. The RNA sequence is complementary to the DNA template strand, with A, T, C, and G in DNA corresponding to U, A, G, and C in RNA. Transcription continues until a termination signal is encountered, which is crucial due to the proximity of adjacent genes in bacteria. Termination sequences are transcribed into RNA, forming a hairpin secondary structure held by hydrogen bonds.  There are two types of transcription termination in bacteria, both involving hairpin structures. Intrinsic termination, which accounts for about 80% of E. coli transcripts, relies on GC-rich sequences forming a stable hairpin followed by a string of uracil residues. The GC-rich hairpin causes RNA polymerase to stall, and the weak interaction between U bases in RNA and A bases in DNA leads to dissociation of RNA polymerase and release of the transcript. Rho-dependent termination is a bacterial transcription termination mechanism used for about 20% of genes in E. coli. This mechanism relies on the termination factor rho (ρ) and a termination sequence that forms a hairpin structure in the transcript. Rho is a large hexameric protein with RNA helicase activity, capable of dissociating RNA secondary structures and DNA/RNA interactions. Rho binds to a specific sequence on the transcript called the rho utilization site (rut) as soon as it is transcribed. Rho moves along the transcript toward the 3' end, chasing RNA polymerase. When RNA polymerase reaches the hairpin structure encoded by the termination sequence, it pauses, allowing Rho to catch up. Rho's RNA helicase activity enables it to move through the hairpin and cause the dissociation of RNA polymerase by breaking the hydrogen bonds between the DNA template and the transcript. 13.10 Transcription in Eukaryotes Differs from Bacterial Transcription in Several Ways Transcription in eukaryotes occurs in the nucleus, requiring mRNA to move to the cytoplasm for translation, unlike in bacteria where transcription and translation can occur simultaneously. Eukaryotes use three different RNA polymerases for transcription, whereas bacteria use only one. Chromatin remodeling is necessary in eukaryotes to uncoil DNA and make it accessible for transcription. Eukaryotic transcription initiation involves multiple general transcription factors (GTFs) and interactions with cis-acting sequences like promoters, enhancers, and silencers, unlike the simpler bacterial system. Termination of transcription in eukaryotes involves sequence-specific cleavage of the transcript, which is more complex than the hairpin structure formation in bacteria. Eukaryotic pre-mRNAs undergo processing to become mature mRNAs, including the addition of a 5' cap, a 3' tail, and the removal of non-coding sequences. Initiation of Transcription in Eukaryotes Eukaryotic RNA polymerase exists in three distinct forms, each larger and more complex than the single form found in bacteria. Yeast and human RNA polymerase II enzymes consist of 12 subunits. The three forms of eukaryotic RNA polymerase share certain protein subunits but transcribe different types of genes. RNA Polymerases in Eukaryotes RNA polymerases I and III transcribe tRNAs and rRNAs, essential for protein synthesis in all cells, while RNA polymerase II (RNAP II) transcribes protein-coding genes and is highly regulated. RNAP II activity is controlled by cis-acting regulatory elements and trans-acting transcription factors. Four types of cis-acting DNA elements regulate RNAP II transcription initiation: - Core promoter: Includes the transcription start site and determines RNAP II binding and transcription initiation. - Proximal-  promoter element: Located upstream of the start site, modulates transcription levels. - Enhancers: Increase transcription efficiency or rate. - Silencers: Decrease transcription efficiency or rate. The TATA box, a core promoter element, is located about 30 nucleotide pairs upstream from the transcription start site and has a consensus sequence TATA(AT). Enhancers and silencers can be located at various distances from the gene and modulate transcription from a distance. Trans-acting factors, or transcription factors, facilitate RNAP II binding and transcription initiation. General transcription factors (GTFs) are required for all RNAP II-mediated transcription and include TFIIA, TFIIB, and TFIID, which binds directly to the TATA box. Transcriptional activators and repressors bind to enhancers and silencers, regulating transcription initiation by influencing the assembly of pre-initiation complexes and the transition to full transcription elongation. During transcription, the enzyme forms a complex with about 40 base pairs of DNA and 18 residues of the growing RNA chain. The earliest synthesized RNA exits through a groove in the enzyme, under a structure called the lid. Ribonucleoside triphosphates (NTPs) enter the enzyme complex through a pore at the bottom. Unlike bacteria, eukaryotic transcription does not have a specific termination sequence; RNAP II often transcribes beyond the eventual 3' end of the mRNA. Transcription is terminated when the polyadenylation signal sequence (AAUAAA) is incorporated, leading to cleavage of the transcript 10-35 bases downstream. Cleavage destabilizes RNAP II, causing the clamp to open and release both DNA and RNA, completing the transcription cycle. The transcription cycle involves an initial unstable DNA-enzyme complex, stabilization during elongation, and instability again at termination. Kornberg's research on transcription earned him the Nobel Prize in Chemistry in 2006. Processing Eukaryotic RNA: Caps and Tails In bacteria, DNA is transcribed into mRNA, which is then directly translated into an amino acid sequence. Eukaryotic mRNAs undergo significant alterations before being transported to the cytoplasm for translation. By 1970, research by James Darnell and others revealed that eukaryotic mRNA is initially transcribed as a larger precursor molecule, known as pre-mRNA. This pre-mRNA must be processed in the nucleus to become mature mRNA before it can be translated in the cytoplasm. Eukaryotic RNA transcripts destined to become mRNAs undergo a posttranscriptional modification at the 5' end, where a 7-methylguanosine (m7G) cap is added. The 5' cap, discovered by Aaron Shatkin and James Darnell, is added shortly after RNA synthesis begins and is crucial for RNA processing within the nucleus. The cap stabilizes mRNA by protecting the 5' end from nuclease attack, facilitates mRNA transport from the nucleus to the cytoplasm, and is required for the initiation of translation. Chemically, the cap is a guanosine residue with a methyl group (CH_3) at position 7 of the base, connected to the initial ribonucleotide of the RNA by a unique 5'-to-5' triphosphate bridge. Some eukaryotes also add a methyl group at the 2'-carbons of the ribose sugars of the first two ribonucleotides. At the 3' end of mRNAs, a poly-A tail consisting of up to 250 adenylic acid residues is added after cleavage of the transcript 10-35 ribonucleotides downstream of the AAUAAA polyadenylation signal sequence. Poly-A polymerase catalyzes the addition of the poly-A tail to the free 3'-OH group at the end of the transcript. The poly-A tail is found at the 3' end of almost all eukaryotic mRNAs, except for those encoding histone proteins. The AAUAAA signal sequence is essential for adding the poly-A tail; mutations in this sequence prevent poly-A tail addition, leading to rapid degradation of the RNA transcript by nucleases.  The poly-A binding protein binds to poly-A tails, preventing degradation and aiding in mRNA export from the nucleus and translation. Poly-A tails are also present in bacterial and archaeal mRNAs, but they are generally shorter and found on fewer mRNA molecules. In bacteria, poly-A tails are associated with mRNA degradation rather than protection. 13.11 The Coding Regions of Eukaryotic Genes Are Interrupted by Intervening Sequences Called Introns The primary mRNA transcript, or pre-mRNA, is often longer than the mature mRNA in eukaryotes. In 1977, research by Phillip Sharp and Richard Roberts revealed that genes of animal viruses contain internal nucleotide sequences that do not encode for amino acids in the final protein product. These noncoding internal sequences, known as introns, are present in pre-mRNAs but are removed during RNA processing to produce mature mRNA. The sequences that are retained in the mature mRNA and expressed are called exons. The process of removing introns from a pre-mRNA and joining together exons is called RNA splicing. Introns in eukaryotic genes are identified by comparing DNA sequences with mRNA sequences and correlating them with amino acid sequences. Common sequences at intron/exon boundaries allow for accurate identification of introns using genomic DNA and computational tools. Genes vary significantly in size, mRNA size, and the number of introns they contain. In Saccharomyces cerevisiae, 283 out of approximately 6000 protein-coding genes contain introns. In humans, about 94% of protein-coding genes contain introns, with an average of nine exons and eight introns per gene. Most mammalian genes contain introns, but exceptions include genes coding for histones and interferon, which have no introns. The pro−α−2(1) collagen gene is an example of a gene with a high number of introns, containing 51 introns. RNA splicing must be highly precise to avoid errors in mature mRNA. There is a significant difference between the length of a gene and the length of the final mRNA after splicing. For example, only 13% of the collagen gene and 8% of the albumin gene consist of exons in the mature mRNA. The dystrophin gene, the largest known human gene, retains less than 1% of its sequence in the mRNA. Why Do Introns Exist? Introns allow for alternative splicing, enabling a single gene to produce multiple protein products by combining different exons, thus increasing the diversity of proteins encoded by the genome. The exon/intron structure facilitates exon shuffling, which can lead to the evolution of new genes by incorporating exons into existing genes. Some introns contain noncoding RNAs, such as microRNAs (miRNAs), which are processed from excised introns and play roles in regulating gene expression. Introns can regulate transcription by containing cis regulatory elements like enhancers and silencers that modulate the transcriptional activity of genes. Splicing Mechanisms: Self-Splicing RNAs Introns are excised and exons are spliced back together through mechanisms that vary across different classes of transcripts and organelles like mitochondria and chloroplasts.  The simplest mechanism involves two steps: cutting the intron at both ends by an endonuclease and joining the exons by a ligase, as seen in bacterial tRNAs. In higher eukaryotes, the excision of introns in tRNAs, rRNAs, and pre-mRNAs is more complex. Eukaryotic introns are categorized into groups based on their splicing mechanisms. Group I introns, found in rRNA primary transcripts, can self-excise without external enzymes. This process results in the intron being spliced out and the exons being ligated to form mature RNA. Self-excision of group I introns occurs in preliminary transcripts for mRNAs, tRNAs, and rRNAs in bacteria, lower eukaryotes, and higher plants. Group II introns are involved in the removal of introns from primary mRNA and tRNA transcripts in mitochondria and chloroplasts. Splicing of group II introns involves two autocatalytic reactions, similar to group I introns. Group II introns are found in fungi, plants, protists, and bacteria. Splicing Mechanisms: The Spliceosome Nuclear-derived protein-coding pre-mRNA introns are much larger and more plentiful than group I and II introns, often exceeding 500,000 nucleotides. Their removal requires a complex mechanism involving the spliceosome, a large molecular complex (40S in yeast and 60S in mammals). Spliceosomal introns are removed by the spliceosome, which includes small nuclear RNAs (snRNAs) and small nuclear ribonucleoproteins (snRNPs). snRNAs, ranging from 80 to 400 nucleotides and rich in uridine residues, are named U1, U2, U3, U4, U5, and U6. snRNPs are complexes of snRNAs and proteins, named after the specific snRNAs they contain (e.g., U2 snRNP contains U2 snRNA). The spliceosome envelops the RNA being spliced, with snRNPs as part of this large structure. Spliceosomal introns have specific nucleotide sequences at their ends: a GU dinucleotide at the 5' end (splice donor sequence) and an AG dinucleotide at the 3' end (splice acceptor sequence). These sequences, along with other consensus sequences, attract specific snRNAs of the spliceosome. The U1 snRNA has a sequence complementary to the 5' splice donor sequence, facilitating the initial binding step in spliceosome formation. Splicing begins after the addition of other snRNPs (U2, U4, U5, and U6). Two transesterification reactions occur during RNA splicing. The first involves an adenine (A) residue within the branch point region of the intron. The 2'-OH of this A residue attacks the phosphodiester bond at the 5' splice site, forming a 5'-to-2' bond between the 5' end of the intron and the branch point A residue. The second transesterification reaction involves the 3'-OH of the upstream exon attacking the phosphodiester bond at the 3' splice site, excising the intron and joining the two exons. The excised intron forms a lariat structure due to the 5'-to-2' bond created in the first reaction. After the second reaction, snRNPs are released, and the intron is typically degraded by nucleases. The 3D structure of the U2/U6 snRNP complex is similar to that of group II self-splicing introns, indicating that group II introns are likely the evolutionary ancestors of the modern spliceosome. RNA splicing can regulate gene expression in eukaryotes. Alternative splicing of introns in nuclear pre- mRNAs from the same gene can produce different collections of exons in mature mRNAs, resulting in related proteins called isoforms. Alternative splicing increases the diversity of proteins that can be produced from a single gene, enhancing the functional complexity of an organism's genome. 13.12 RNA Editing May Modify the Final Transcript  RNA editing is a form of posttranscriptional RNA processing where the ribonucleotide sequence of a pre- mRNA is altered before translation, resulting in a mature RNA sequence that differs from the DNA template. There are two main types of RNA editing: - Substitution editing: Individual nucleotide bases are chemically modified. This type is found in some nuclear-derived eukaryotic RNAs and is common in mitochondrial and chloroplast RNAs in plants. - Insertion/deletion editing: Nucleotides are added or removed from the RNA sequence. This type is used by organisms like Physarum polycephalum and Trypanosoma. In Trypanosoma, insertion/deletion editing is extensive, with uridines added to transcripts forming up to 60% of the coding sequence, often creating the initiation codon and aligning the reading frame. Guide RNAs (gRNAs) direct insertion/deletion editing in trypanosomes by base-pairing with pre-edited mRNAs to guide the editing machinery. Substitutional editing in mammalian nuclear-encoded mRNA transcripts includes the editing of apolipoprotein B (ApoB) mRNA in human intestinal cells. A single C-to-U change converts a CAA glutamine codon into a UAA stop codon, producing a shorter polypeptide. The editing of ApoB mRNA involves a protein complex including APOBEC-1, which binds to a mooring sequence on the pre-mRNA just downstream of the editing site.