Chapter 21
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Transcript of Chapter 21
© 2012 Pearson Education, Inc.
Lectures byKathleen Fitzpatrick
Simon Fraser University
Chapter 21
Gene Expression I: The Genetic Code and Transcription
© 2012 Pearson Education, Inc.
The Genetic Code and Transcription
• The coded information of DNA is used to guide RNA production and the subsequent translation into protein
• The synthesis of RNA molecules is called transcription
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The Directional Flow of Genetic Information
• DNA serves as a template for the synthesis of an RNA molecule which then directs the synthesis of a protein product
• Sometimes the RNA itself is the final product
• The principle of directional information flow from DNA to RNA to protein is the central dogma of molecular biology
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Transcription and translation
• Transcription refers to RNA synthesis using DNA as a template
• Translation is the synthesis of protein using the information in the RNA
• Messenger RNA, mRNA, is RNA that is translated into protein
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Additional types of RNA
• Ribosomal RNA, rRNA, is an integral component of the ribosome
• Transfer RNA, tRNA, molecules serve as intermediaries, bringing amino acids to the ribosome
• Both function during translation
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Refinements of the central dogma
• There are exceptions to the central dogma
• For example, there are RNA viruses that carry out reverse transcription, using RNA as a template for DNA synthesis
• Other viruses produce RNAs from an RNA template
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The Genetic Code
• The relationship between the DNA base sequence and the linear order of amino acids in the protein products is based on a set of rules known as the genetic code
• They detected a link between gene mutations and proteins
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Mutants and metabolic pathways
• Beadle and Tatum grew mutants on minimal medium with metabolic precursors of a particular amino acid or vitamin
• They determined which precursors allowed the growth of each mutant
• They were able to infer that each mutation disabled a single enzymatic step of a metabolic pathway, the one-gene-one-enzyme hypothesis
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Most Genes Code for the Amino Acid Sequences of Polypeptide Chains
• Linus Pauling studied the inherited disease sickle-cell anemia, in which the red blood cells assume a sickle shape
• He analyzed hemoglobin using electrophoresis and found that hemoglobin of sickle cells migrated differently from normal hemoglobin
• Vernon Ingram used the protease trypsin to cleave hemoglobin into fragments and then examined the peptides
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Sickle-cell hemoglobin differs from normal hemoglobin
• Ingram found just one amino acid difference between normal and sickle-cell hemoglobin
• The sickle-cell hemoglobin has a valine instead of a glutamic acid; a neutral amino acid instead of a negatively charged one
• This changed the one-gene-one-enzyme hypothesis; hemoglobin is not an enzyme
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A refined hypothesis
• The new hypothesis was refined to the one-gene-one-polypeptide theory: the nucleotide sequence of a gene determines the amino acid sequence of a polypeptide chain
• Charles Yanofsky showed that mutations in the bacterial tryptophan synthase gene corresponded to changed amino acids in the polypeptide
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Gene function is complicated
• Most eukaryotic genes contain noncoding sequences among the coding regions of the gene
• Coding sequences can be read in various combinations, each coding for a unique polypeptide chain; this is called alternative splicing
• Some types of genes encode functional RNAs
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The Genetic Code Is a Triplet Code
• There are four DNA bases and 20 amino acids
• A doublet code, in which two bases specify a single amino acid, is inadequate as only 16 combinations are possible
• A triplet code, in which combinations of three bases specify amino acids, would have 64 possible combinations, more than enough for all 20 amino acids
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Frameshift mutations
• The gene is written in a language of three-letter words
• Inserting or deleting a nucleotide causes the rest of the sequence to be read out of phase—this is a shift in the reading frame
• Mutations that cause insertion or deletion of a nucleotide are thus called frameshift mutations
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The Genetic Code Is Degenerate and Nonoverlapping
• There are 64 combinations of nucleotide triplets and only 20 amino acids
• This means the genetic code is degenerate, meaning that a particular amino acid can be specified by more than one triplet
• It is also nonoverlapping; the reading frame advances three nucleotides at a time
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The genetic code
• Although the genetic code is always nonoverlapping, there are cases where a segment of DNA is translated in more than one reading frame
• E.g., some viruses with very small genomes have overlapping genes, and some bacteria have genes that slightly overlap
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Messenger RNA Guides the Synthesis of Polypeptide Chains
• The genetic code refers to the order of nucleotides in the mRNA molecules that direct protein synthesis
• mRNA is transcribed from DNA similarly to how DNA is replicated, but with two differences
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Differences between mRNA synthesis and DNA replication
• In mRNA synthesis, only one DNA strand is copied, called the template strand; the other strand is called the coding strand because it is similar to the mRNA sequence
• In mRNA synthesis, a uracil base (U) is used instead of thymine
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Cell-free systems
• Nirenberg and Matthei pioneered the use of cell-free systems for studying protein synthesis
• They decided to add synthetic RNAs of known sequence to the cell-free system
• They used polynucleotide phosphorylase to make synthetic RNA molecules of predictable base composition
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Working out the genetic code
• When a single ribonucleotide is used to make RNA the RNA is called a homopolymer
• When poly (U), but not other homopolymers, was added to the cell-free system, a large amount of phenylalanine was incorporated, suggesting that UUU specifies phenylalanine
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The Codon Dictionary Was Established Using Synthetic RNA Polymers and Triplets
• RNA triplets, called codons, are read by the transcriptional machinery
• Further homopolymer experiments showed AAA codes for lysine, and CCC codes for proline
• Copolymers were tested (containing a mixture of two nucleotides) but it was difficult to be sure which codon specified each amino acid
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A different approach
• Khorana used an approach with one important difference—he synthesized the RNA molecules in an alternating sequence
• This sort of copolymer has only two codons, e.g., UAUAUAUA UAU and AUA, and Khorana could narrow the codon assignments to either tyrosine or isoleucine
• Eventually, these experiments allowed assignment of all the codons
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Of the 64 Possible Codons in Messenger RNA, 61 Code for Amino Acids
• All 64 codons are used in the translation of mRNA
• 61 of them specify the addition of specific amino acids to a growing polypeptide chain
• One of them, AUG, plays a role as a start codon
• The remaining 3 (UAA, UAG, UGA) are stop codons, which terminate polypeptide synthesis
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The genetic code is unambiguous and degenerate
• Every codon has one meaning only, the genetic code is unambiguous
• It is also degenerate—many of the amino acids are specified by more than one codon
• With a degenerate code, most mutations cause codon changes and a changed amino acid
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The Genetic Code Is (Nearly) Universal
• Except for a few cases all organisms use the same basic genetic code
• In the case of mitochondria, and a few bacteria, the genetic code differs in several ways
• E.g., AGA is a stop codon in mammalian mitochondria and in some organisms codons specify nonstandard amino acids
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Transcription in Bacterial Cells
• The fundamental principles of transcription were first elucidated in bacteria, where molecules and mechanisms are relatively simple
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Transcription Is Catalyzed by RNA Polymerase, Which Synthesizes RNA Using DNA as a Template
• Transcription is carried out by the enzyme RNA polymerase
• Bacteria have a single kind of RNA polymerase to synthesize all three classes of RNA—mRNA, tRNA, and rRNA
• The RNA polymerase of E. coli has two two subunits, and a dissociable sigma () factor
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Transcription Involves Four Stages: Binding, Initiation, Elongation, and Termination
• The DNA that gives rise to one RNA molecule is called the transcription unit
• Transcription begins when RNA polymerase binds to a promoter sequence (1) triggering local unwinding of the double helix
• RNA polymerase then initiates synthesis of RNA using one DNA strand as a template (2)
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Steps of RNA synthesis (continued)
• After initiation the RNA polymerase moves along the DNA template, unwinding the helix and elongating the RNA (3)
• Eventually the enzyme transcribes a termination signal which stops RNA synthesis and causes release of the RNA and dissociation of the polymerase (4)
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Binding of RNA Polymerase to a Promoter Sequence
• RNA polymerase binds to a DNA promoter site, a sequence of several dozen base pairs that determines where RNA synthesis will start
• The terms upstream and downstream refer to sequences located toward the 5 or 3 end of the transcription unit, respectively
• The promoter is upstream of the transcribed sequence
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Initiation of RNA Synthesis
• Initiation of RNA synthesis takes place once the DNA is unwound
• One of the DNA strands serves as a template for RNA synthesis, using incoming NTPs that are complementary to the template strand
• RNA polymerase catalyzes the formation of a phosphodiester bond between the NTPs
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Elongation of the RNA Chain
• Chain elongation continues as RNA polymerase moves along the DNA molecule
• The RNA is elongated in the 5 to 3 direction, with each new nucleotide added to the 3 end
• As the polymerase moves along the DNA strand, the double helix ahead of the polymerase is unwound and the DNA behind it is rewound into a double helix
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RNA polymerases have exonuclease activity
• When an incorrect nucleotide is incorporated, the polymerase backs up slightly and the incorrect nucleotide and the previous one are removed
• This is RNA proofreading; occasional errors in RNA molecules are not as critical as errors in DNA replication
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Termination of RNA Synthesis
• Elongation of the RNA chain proceeds until the RNA polymerase copies a sequence called the termination signal
• There are two types of termination signals based on whether or not they require a protein called the rho factor
• RNA molecules that terminate without the rho factor contain
a short GC-rich sequence followed by several Us
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Types of termination signal (continued)
• RNA molecules that don’t form the GC-rich hairpin require the rho factor for termination
• The rho factor is an ATP-dependent unwinding enzyme moving along the RNA molecule toward the 3 end and unwinding it from the DNA template as it proceeds
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Transcription in Eukaryotic Cells
• Eukaryotic transcription involves the same four stages as prokaryotic but there are several important differences
– Each of three different RNA polymerases transcribes one or more different classes of RNA
– Eukaryotic promoters are more varied than bacterial ones, some are even located downstream of the gene
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Eukaryotic transcription
• Eukaryotic transcription differs from that of prokaryotes
– RNA polymerases in eukaryotes require additional proteins called transcription factors, some of which must bind before the RNA polymerase can bind
– Protein-protein interactions play a prominent role in eukaryotic transcription
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Eukaryotic transcription (continued)
• Eukaryotic transcription differs from that of prokaryotes
– RNA cleavage is more important than termination of transcription in determining the 3 end of the transcript
– Newly forming RNA molecules undergo RNA processing, chemical modification during and after transcription
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RNA Polymerase I, II and III Carry Out Transcription in the Eukaryotic Nucleus
• There are three RNA polymerases in the nucleus designated RNA polymerases I, II, and III
• These differ in their location in the nucleus and the types of RNA they synthesize
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The RNA polymerases
• RNA polymerase I, in the nucleolus, synthesizes an RNA molecule that is a precursor for three types of rRNA
• RNA polymerase II is found in nucleoplasm and synthesizes mRNA; the molecules are found in clusters called transcription factories, where active genes congregate to be transcribed
• RNA polymerase II is very sensitive to -amanitin, unlike polymerase I
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The RNA polymerases (continued)
• RNA polymerase III, in the nucleoplasm, synthesizes a variety of small RNAs including tRNA, and the 5S rRNA
• It is sensitive to -amanitin but only at higher levels than polymerase II
• All three polymerases are large, and composed of multiple polypeptide subunits
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Three Classes of Promoters Are Found in Eukaryotic Nuclear Genes, One for Each Type of RNA Polymerase
• Eukaryotic promoters are varied, but can be grouped into three categories
• The promoter used by RNA polymerase I has two parts
• The core promoter is the smallest set of DNA sequences that initiates transcription
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The upstream control element
• The core promoter is sufficient for initiation of transcription
• However, transcription occurs more efficiently in the presence of an upstream control element, a fairly long sequence similar to the core promoter
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The promoter for RNA polymerase II
• At least four types of DNA sequences are involved in core promoter function
• 1. A short initiator sequence surrounds the transcription startpoint
• 2. The TATA box, a consensus sequence of TATA followed by 2-3 As, is located about 25 nucleotides upstream of the startpoint
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The promoter for RNA polymerase II (continued)
• Four types of DNA sequences are involved in core promoter function (continued)
• 3. The TFIIB recognition element (BRE) is located slightly upstream of the TATA box
• 4. The downstream promoter element (DPE) is located about 30 nucleotides downstream from the startpoint
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Additional control elements
• Core promoters are only capable of driving a basal (low) level of transcription
• Additional short sequences upstream (upstream control elements) improve the promoter’s efficiency
• Some are common to many different genes, e.g., the CAAT box and the GC box
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Upstream control elements
• The location of upstream control elements varies from gene to gene
• Those within 100–200 nucleotides of the startpoint are called proximal control elements
• Those farther away are called enhancer elements
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Promoters for RNA polymerase III
• RNA polymerase III uses promoters that are entirely downstream of the startpoint
• In both 5S RNA and tRNA the promoters are different but both consensus sequences fall into two blocks of about 10 bp each
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General Transcription Factors Are Involved in the Transcription of All Nuclear Genes
• A general transcription factor is always required for RNA polymerase binding to promoters
• Eukaryotes have many such factors, called TFs, that bind the promoter in a defined order starting with TFIID
• Eventually a large complex of proteins forms a preinitiation complex on the promoter
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Elongation, Termination, and RNA Cleavage Are Involved in Completing Eukaryotic RNA Synthesis
• After initiation RNA polymerases move along the DNA and synthesize a complementary RNA
• Termination is governed by signals that differ for each type of RNA polymerase
• Transcription by polymerase I is terminated by a protein that recognizes an 18-nucleotide signal in the growing RNA chain
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Termination of transcription
• For RNA polymerase III, termination signals include a short run of Us and no protein factors are required for their recognition
• For RNA polymerase II, transcripts are cleaved at a specific site before transcription ceases
• The cleavage site is 10–35 nucleotides downstream of a AAUAAA sequence in the RNA
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Polyadenylation
• The cleavage site of polymerase II transcripts is also the site for addition of a poly(A) tail
• This is a string of adenine nucleotides added to the 3 end of most eukaryotic mRNAs
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RNA Processing
• A newly produced RNA molecule is called the primary transcript
• It must undergo RNA processing (chemical modification) before it can function in the cell
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Ribosomal RNA Processing Involves Cleavage of Multiple rRNAs from a Common Precursor
• rRNA is the most abundant and stable form of RNA in cells
• Four types of rRNA are distinguished by their different sedimentation rates during centrifugation
• The small ribosomal subunit has one 18S rRNA molecule, whereas the larger has three (28S, 5.8S, and 5S)
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Processing of rRNAs
• The three larger eukaryotic rRNAs are encoded by a single transcription unit, which produces a primary transcript called the pre-rRNA
• The three rRNAs are separated by transcribed spacers
• A series of cleavage reactions remove the spacers, and methyl groups are added to the pre-rRNA
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Ribosome assembly in the nucleolus
• Processing of pre-rRNA is accompanied by assembly of the RNA with proteins to form the ribosomal subunits
• 5S RNA is transcribed by RNA polymerase III in a separate transcription unit with multiple copies in long tandem arrays
• 5S rRNA transcripts require little or no processing
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Transfer RNA Processing Involves Removal, Addition, and Chemical Modification of Nucleotides
• Cells synthesize several dozen kinds of tRNA molecules
• They fold into a secondary structure, most containing four hairpin loops; but some have a fifth region called a variable loop
• tRNAs have a cloverleaf structure, and are synthesized as pre-tRNAs, followed by processing
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The events of processing the pre-tRNA
• At the 5 end a short leader sequence (16 nucleotides) is removed (1)
• At the 3 end, the two terminal nucleotides are removed and replaced with CCA (2)
• About 10–15% of the nucleotides are chemically modified (3)
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Pre-tRNA processing (continued)
• Types of chemical modifications include methylation and creation of unusual bases (dihydrouracil, ribothymine, pseudouridine, inosine)
• An internal 14-nucleotide sequence is removed, though only for a few tRNAs (4)
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Messenger RNA Processing in Eukaryotes Involves Capping, Addition of Poly(A), and Removal of Introns
• Most bacterial RNA is synthesized in a form that is ready for translation with no need for processing
• Because there is no nuclear membrane, bacterial transcripts are translated as they are transcribed
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Transcription and translation in eukaryotes
• Eukaryotic transcripts must be exported from the nucleus to be translated
• Substantial processing occurs in the nucleus before export
• Primary transcripts are often very long, 2,000–20,000 nucleotides, referred to as heterogeneous nuclear RNA (hnRNA)
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Eukaryotic transcripts
• Pre-mRNAs are processed by removal of sequences and addition of 5 caps and 3 tails
• The C-terminal domain of one of the subunits of RNA polymerase II acts as a platform for protein complexes involved in processing
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5 Caps and 3 Poly(A) Tails
• Eukaryotic mRNAs have a modified nucleotide called the 5 cap and the 3 ends have a long stretch of adenines called the poly(A) tail
• The 5 cap is a guanosine that is methylated at position 7 of the purine ring
• It is bound to the RNA molecule by a 5–5 linkage rather than the usual 3–5 bond
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Roles of the 5 cap
• The 5 cap is added soon after transcription is initiated
• The cap contributes to mRNA stability by protecting the RNA from nucleases
• The cap also plays a role in positioning the RNA on the ribosome for initiation of translation
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The poly(A) tail
• The poly(A) tail ranges from 50 to 250 nucleotides long and is added by the enzyme poly(A) polymerase
• A signal, AAUAAA, is located just upstream of the polyadenylation site, and a GU- or U-rich element is located downstream of it
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Function of the poly(A) tail
• The poly(A) tail protects the mRNA from nuclease attack; the length of the tail influences stability
• It is also required for export of the transcript to the cytoplasm
• It may also help ribosomes recognize and bind mRNAs
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The Discovery of Introns
• The precursors for most mRNAs and some rRNAs and tRNAs contain introns, sequences within the primary transcript that are removed
• Experiments demonstrated that eukaryotic gene sequences contain extra DNA that does not appear in the mature RNA
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Exons and introns
• Sequences that appear in the final mRNA were called exons
• Introns are present in most protein coding genes of multicellular eukaryotes
• The size and number of introns varies considerably
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Spliceosomes Remove Introns from Pre-mRNA
• The process of removing introns and joining the exons is RNA splicing
• About 15% of inherited human diseases involve splicing errors; such errors lead to incorrect protein products
• Sequences commonly found at the intron-exon boundaries likely determine the 5 and 3 splice sites
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Splice sites
• Analysis of base sequences of hundreds of different introns revealed that the 5 end of an intron typically starts with GU and terminates with AG at the 3 end
• The sequences immediately adajcent to the 3 and 5 ends of the intron tend to be similar
• One additional sequence near the 3 end of the intron is called the branch point
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The Existence of Introns Permits Alternative Splicing and Exon Shuffling
• In some cases introns are processed to yield functional products
• In few cases introns are translated into proteins
• However most introns are destroyed without serving any obvious function
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Alternative splicing
• The presence of introns allows each gene’s pre-mRNA molecule to be spliced in multiple ways, leading to production of multiple protein products
• This alternative splicing is possible via mechanisms allowing certain splice sites to be activated or skipped
• Regulatory proteins and snoRNAs bind to splicing enhancer or silencer sequences
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Intron functions
• Besides alternative splicing, introns allow the evolution of new protein-coding genes through recombination events
• Recombination between introns produces new combinations of exons—exon shuffling
• It can also produce duplicate copies of exons within a gene, one of which could mutate to a new sequence
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RNA Editing Allows mRNA Coding Sequences to Be Altered• Another type of RNA processing is RNA editing
• Anything from a single nucleotide to hundreds may be inserted, removed, or altered in the mRNA
• Some of the best-studied examples occur in mitochondria of trypanosomes
• Small guide RNAs, encoded by different mitochondria genes, determine the location for the placement of the Us
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Key Aspects of mRNA Metabolism
• Two key aspects of mRNA metabolism are important to understanding mRNA behavior in cells
• mRNAs have a short life span
• mRNAs have the ability to amplify genetic information
• mRNA can be synthesized again and again from a piece of template DNA, providing an opportunity for amplification of genetic information
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Most mRNA Molecules Have a Relatively Short Life Span
• Most mRNA molecules have a high turnover rate (rate at which molecules are degraded and replaced)
• It is measured in terms of half-life, the time required for 50% of the molecules to degrade
• mRNA molecules of eukaryotes have half-lives of several hours to a few days; in bacteria, the half-lives are usually only a few minutes