© 2012 Pearson Education, Inc. Lectures by Kathleen 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Experiments on Neurospora Revealed That Genes Can Code for Enzymes Beadle and Tatum studied the common bread mold, Neurospora crassa, in the early 1940s They detected a link between gene mutations and proteins They used X-ray to generate mutations that were unable to survive on minimal medium though they could grow on complete medium (supplemented)

© 2012 Pearson Education, Inc. Mutants of Neurospora The mutants generated had lost the ability to synthesize certain amino acids or vitamins They could only survive when the missing substances were provided by the supplemented medium They used a variety of supplemented media to deduce exactly which nutrients were missing in a large number of different mutants

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Frameshift Mutations In 1961, Crick, Brenner, and others provided evidence for the triplet code Proflavin (an acridine dye) was used as a mutagen in bacteriophage T4 Some of the mutations induced were revertible; they could be restored to wild type

© 2012 Pearson Education, Inc. The revertible mutations were not a true reversal of the mutation Upon examining the revertants, it was discovered that, rather than a reversal of the original mutation, they contained a second mutation near the first By itself either the original mutation or the revertant will cause a mutant phenotype, but together they “cancel each other out” The phenotype is called “pseudo wild-type”

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Evidence for a Triplet Code Crick and Brenner generated frameshift mutations in T4 phage and found that these could acquire a second mutation that returned the phage to pseudo wild-type When three nucleotides are added or removed, the reading frame does not change, suggesting that nucleotides are read in groups of three

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. RNA polymerase The core enzyme lacks the sigma subunit and can carry out RNA synthesis However the holoenzyme (including all the subunits) is required to ensure initiation at all sites within the DNA molecule The sigma subunit promotes binding of RNA polymerase at promoters, DNA sequences at the beginnings of genes

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Promoters RNA polymerase binding the promoter is mediated by the sigma subunit Promoter sequences were identified by DNA footprinting and electrophoretic mobility shift assays More recently chromatin immunoprecipitation (ChIP) has been used

© 2012 Pearson Education, Inc. Promoters in bacteria DNA promoters differ significantly in bacterial transcription units Enzyme recognition and binding depends on short sequences within the promoter sequences The startpoint is where transcription begins, nearly always a purine

© 2012 Pearson Education, Inc. Other features of promoters in bacteria About 10 bases upstream of the startpoint is the  10 sequence or Pribnow box (TATAAT) About 35 bases upstream of the startpoint (  35) is the  35 sequence (TTGACA) These sequences are evolutionarily conserved but are not identical; TATAAT and TTGACA are consensus sequences

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Types of termination signal RNA molecules that terminate wthout the rho factor contain a short GC-rich sequence followed by several Us The GC region in the RNA forms a hairpin loop pulling the RNA molecule away from the DNA Then the weaker bonds between the Us and the As of the template strand break, releasing the RNA

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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, as well as sensitivity to various inhibitors, such as  -amanitin

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Two types of core promoters The four elements are organized into two general types of core promoters TATA-driven promoters contain an Inr sequence and a TATA box with or without an associated BRE DPE-driven promoters contain DPE and Inr sequences but no TATA box or BRE

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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 tRNA has box A and box B; rRNA has box A and box C

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. General transcription factors TFIIH possesses helicase activity that unwinds DNA and protein kinase activity that phosphorylates RNA polymerase II This releases RNA polymerase II from the transcription factors so it can begin RNA synthesis TFIID recognizes and binds DNA due to its TATA binding protein (TBP) subunit

© 2012 Pearson Education, Inc. Other proteins needed for transcription Besides general transcription factors and RNA polymerase II, several other kinds of proteins are needed Some open chromatin structure to make DNA accessible to RNA polymerase, others are regulatory factors that bind upstream control elements and recruit coactivator proteins

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. Processing of rRNAs (continued) The main site of methylation is the 2-hydroxyl group of ribose, but a few bases are methylated too Methylation and cleavage are guided by small nucleolar RNAs (snoRNAs) Methylation may protect certain sequences on the pre-rRNA from cleavage

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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)

© 2012 Pearson Education, Inc. Eukaryotic transcripts hnRNA is a mixture of mRNA molecules and their precursors, pre-mRNA 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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 This was demonstrated by R looping

© 2012 Pearson Education, Inc. R looping Single-stranded RNA and double-stranded DNA are hybridized under conditions that favor heteroduplexes, RNA-DNA hybrid molecules DNA that is not hybridized to RNA is displaced as a single-stranded loop, easily observed with electron microscopy Some genes contained multiple loops

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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 adacent 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

© 2012 Pearson Education, Inc. The spliceosome Intron removal is catalyzed by large molecular complexes called spliceosomes, consisting of five types of RNA and many proteins Spliceosomes assemble on transcripts from a group of smaller RNA-protein complexes called snRNPs (small nuclear riboprotein complexes), each containing one or two snRNA molecules (small nuclear RNA)

© 2012 Pearson Education, Inc. Spliceosome assembly Spliceosomes are assembled by sequential binding of snRNPs to pre-mRNA The first step is the binding of a snRNP called U1, which contains an RNA that can base pair with the 5 splice site A second snRNP called U2 binds to the branch- point sequence

© 2012 Pearson Education, Inc. Spliceosome assembly (continued) Finally a group of snRNPs (U4/U6 and U5) brings the ends of the intron together to form a mature spliceosome The pre-mRNA is cleaved at the 5 splice site, which is joined to an adenine residue located at the branch-point sequence The resulting structure is called a lariat

© 2012 Pearson Education, Inc. Spliceosome assembly (continued) After the lariat forms, the 3 splice site is cleaved and the two ends of the exon are joined together A multiprotein complex called an exon junction complex (EJC) is deposited near the boundary of each newly formed exon-exon junction

© 2012 Pearson Education, Inc. A second class of introns A second class of introns with AU and AC at their 5 and 3 splice sites, has been identified These are often excised by a second type of spliceosome that differs in snRNP composition In both cases, the snRNA molecules in the spliceosomes are directly involved in splice-site recognition, spliceosome assembly, and splicing

© 2012 Pearson Education, Inc. Some Introns Are Self-Splicing A few types of genes have self-splicing RNA introns These have RNAs that carry out splicing without the help of any proteins; the intron itself catalyzes the process RNA molecules that function as catalysts are called ribozymes

© 2012 Pearson Education, Inc. Group I and group II introns The first class of self-splicing introns is called Group I introns These are excised in the form of linear RNA fragments Group II introns are excised as lariats

© 2012 Pearson Education, Inc. The Existence of Introns Permits Alternative Splicing and Exon Shuffling In some cases introns are processed to yield functional products In a few cases introns are translated into proteins However most introns are destroyed without serving any obvious function

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. RNA editing in trypanosome mitochondria Editing involves insertion and deletion of multiple uracil nucleotides at numerous points in the mRNA Small guide RNAs, encoded by different mitochondria genes, determine the location for the placement of the Us In some other examples, Cs are converted to Us and vice versa

© 2012 Pearson Education, Inc. Other examples of editing MicroRNAs are another class of RNA that can be edited DNA can also be edited, e.g., eukaryotes have a DNA-editing enzyme called APOBEC3G that inactivates retroviruses Viruses such as HIV produce a protein called Vif, which destroys APOBEC3G

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. 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

© 2012 Pearson Education, Inc. The Existence of mRNA Allows Amplification of Genetic Information mRNA can be synthesized again and again from a piece of template DNA, providing an opportunity for amplification of genetic information

© 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University Chapter 21 Gene Expression I: The Genetic Code and Transcription.

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© 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University Chapter 21 Gene Expression I: The Genetic Code and Transcription.

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