Bio-Synthesis of RNA Processing and Modification of RNA Protein Engneering

1

RNA Synthesis, Processing and ModificationProtein Synthesis and Genetic Code

2Joko SetyonoBiochemistry DepartmentMedical and Health Sciences Faculty of UNSOEDab7616sa@yahoo.com

3MenuWhat are the different kinds of RNA?

How are genes transcribed into RNA?

How are mRNAs prepared (matured) for translation?

How are mRNAs translated into proteins?

What is the structural basis of protein function?and how mutations can alter the function of proteins? 4OverviewRNA is transcribed by RNA polymerase and associated enzymatic machinery, following the rules of base pairing from the template strand of DNA.Most genes code for protein or for functional RNAs used in protein synthesis and other functions.The nucleotide sequence of the gene determines the order of amino acids in a protein, which determines shape, size, and protein function.mRNA is translated in groups of three nucleotides (codon) at the ribosome through pairing of tRNA anticodon with the mRNA codon.Change in nucleotide sequence (mutation) may cause change in amino acid sequence, altering function.5

6

7

8

9

10

11 The Central Dogma of Molecular Biology: DNA------>RNA------>protein

The central dogma concerns the flow of biological information: DNA is a self-replicating molecule containing genetic information that can be transcribed into an RNA message that can be translated into a polypeptide (protein).

12

Central DogmaAre all RNA translated to protein?13Synthesis of three types of informational molecules: DNA,RNA and Protein. Note that only one strand of the DNA is transcribed into RNA.The Central Dogma of molecular biology

1. RNARNA Structure & Function RNA structure Levels of organization Bonds & energetics

RNA types & functions Genomic information storage/transfer Structural Catalytic Regulatory

RNA structure: 3 levels of organization

Covalent & non-covalent bonds in RNA

Primary: Covalent bonds

Secondary/Tertiary Non-covalent bonds H-bonds (base-pairing) Base stacking Common structural motifs in RNA

Helices

Loops Hairpin Interior Bulge Multibranch

Pseudoknots

RNA functions Storage/transfer of genetic information

Structural

Catalytic

Regulatory

RNA functions Storage/transfer of genetic information

Genomes many viruses have RNA genomessingle-stranded (ssRNA)e.g., retroviruses (HIV)double-stranded (dsRNA)

Transfer of genetic information mRNA = "coding RNA" - encodes proteins

RNA functionsStructural e.g., rRNA, which is major structural component of ribosomes (Gloria Culver, ISU) BUT - its role is not just structural, also:

Catalytic RNA in ribosome has peptidyltransferase activity Enzymatic activity responsible for peptide bond formation between amino acids in growing peptide chain Also, many small RNAs are enzymes "ribozymes" (W Allen Miller, ISU)RNA functionsRegulatory

Recently discovered important new roles for RNAs In normal cells: in "defense" - esp. in plants in normal developmente.g., siRNAs, miRNAAs tools: for gene therapy or to modify gene expression RNAi (used by many at ISU: Diane Bassham,Thomas Baum, Jeff Essner, Kristen Johansen, Jo Anne Powell-Coffman, Roger Wise, etc.) RNA aptamers (Marit Nilsen-Hamilton, ISU)The origin and structure of RNATranscription: copying nucleotide sequence of DNA into RNAforms RNA transcriptDNA may be transcribed multiple timesRNAsingle-stranded polynucleotidecontains ribose sugarcontains the pyrimidine uracil (U) (CUT Py)hydrogen bonds with A5 and 3 ends critically importantMay fold back on itself and adopt a specific and stable secondary structure

2 major classes of RNAInformational RNA = protein-coding = messenger RNAprimary transcript in prokaryotesprocessed transcript in eukaryotes5 and 3 end modificationintron removalgenerally transcribed by RNA polymerase IItranslated into amino acid sequence

Functional RNA = non-coding RNAalso encoded by genes but no coding potential for amino acid sequencetranscribed by various RNA polymerases (I, II, III)Some are among the most highly conserved genes in nature (rRNA)The RNA Content of the Cell Total RNANon-coding RNA96% of totalCoding RNA4% of totalPre-mRNA(hnRNA)mRNAPre-rRNAPre-tRNAsnRNAsnoRNAscRNAtmRNA andvariousother typesrRNAtRNA All organisms Bacteria only Eukaryotes onlyRNA types & functions Types of RNAsPrimary Function(s)mRNA - messenger translation (protein synthesis) regulatoryrRNA - ribosomal translation (protein synthesis) t-RNA - transfer translation (protein synthesis)hnRNA - heterogeneous nuclearprecursors & intermediates of mature mRNAs & other RNAsscRNA - small cytoplasmic signal recognition particle (SRP)tRNA processing snRNA - small nuclear snoRNA - small nucleolarmRNA processing, poly A addition rRNA processing/maturation/methylationregulatoryRNAs (siRNA, miRNA, etc.)regulation of transcription and translation, other??Many types of functional non-coding RNA including:tRNA: transfer RNA, transport amino acid to ribosomerRNA: ribosomal RNA, structural and catalytic component of ribosomessnRNA: small nucleolar RNA, structural and catalytic component of spliceosome snRNPsscRNA: small cytoplasmic RNA, direct protein traffic in cytoplasmmiRNA: micro RNA, involved in post-transcriptional gene regulation

Svedberg Coefficient (S) sedimentation behavior in a centrifugal field. Sedimentation behavior depends on mass, density and shapeAlsosnRNAs (small nuclear RNAs)miRNAs (microRNA & interfering RNA, RNAi)2. TranscriptionBasic principles of transcriptionTranscription is synthesis of RNA using a DNA templateThree Key differences between DNA and RNA:RNA contains the sugar ribose instead of deoxyriboseRNA contains the base uracil instead of thymineExcept in certain viruses RNA is not a doublestranded moleculeThree major types of RNA - all products of transcription of DNA:Messenger RNA (mRNA)Transfer RNA (tRNA)Ribosomal (rRNA)31

The function of RNA polymerase is to copy one strand of duplex DNA into RNA.

37

Transcription is the process by which nucleotide sequences in DNA are copied to a complementary copy of messenger RNA38Transcription: who are the players?

RNA Polymerases: Basic function and featuresadds free ribonucleotides to growing RNA strand at 3 end (thus direction for transcription is 5 -> 3) Core RNA polymerase: huge protein complex made of multiple subunits, each encoded by different genesRequires many other factors, either other proteins or co-factors (in particular for binding to DNA) = functional complex is called RNA Pol holoenzymeUnlike DNA pol, RNA pol does not require a primer to initiate synthesis

RNA polymeraseBinds the DNA templateBinds the NTPKeeps the core togetherTemporary partner recognizes the promoter of the gene to be transcribed. First mechanism for gene regulation.Different s subunits recognize different promoters with different efficiencies!41

RNA polymerase is the enzyme that copies DNA into a complementary copy of RNA.

The enzyme uses DNA as a template and since it catalyzes the addition of ribonucleotides in 5'-->3' direction, it reads its template DNA in the 3'-->5' direction.RNA polymerasesProkaryotes: single RNA polymeraseEukaryotes: three RNA polymerasesRNA pol I transcribes rRNA genesRNA pol II transcribes protein- encoding genesprimary transcript will be processedRNA pol III transcribes tRNA genes and 5S rRNA genes

43

Promoters

Promoters are specific sites on DNA that RNA polymerase first binds to to initiate the transcription of a gene.44

Sigma factors

Sigma factors are one component of the multicomponent RNA polymerase enzyme that allow the RNA polymerase to recognize the initiation (promoter) site.45

Transcription Terminators

Transcription terminators are sequences of nucleotide bases at the end of the gene that signal termination of transcription.

Transcription stepsInitiationat 5 end of genebinding of RNA polymerase to promoterunwinding of DNAElongationaddition of nucleotides to 3 endrules of base pairingrequires Mg2+ energy from NTP substratesTerminationat 3 end of geneterminator loop (prokaryote) or processing enzymecoding region5UTR3UTR48

49

How does transcription initiate? Four stages of Transcription50

How does transcription terminate?Intrinsic terminators

51

A rho-dependent terminator has a sequence rich in C and poor in G preceding the actual site(s) of termination. 52

Rho factor pursues RNA polymerase along the RNA and can cause termination when it catches the enzyme pausing at a rho-dependent terminator. 53Transcription in EukaryotesRNA polymerase I transcribes rRNA RNA polymerase II transcribes mRNARNA polymerase III transcribes tRNA and other small RNAs.

3. RNA maturation/processing57Major RNA processing mechanisms in eukaryotesribosomal RNA (RNA polymerase I transcripts)(i)methylation(ii)nucleolytic cleavage messenger RNA (RNA polymerase II transcripts) (i)5-capping(ii)3-cleavage and polyadenylation(iii)removal of introns (nuclear splicing)

transfer RNA (RNA polymerase III transcripts)(i)5- and 3-cleavage(ii)CCA addition(iii)base modification(iv)tRNA splicingEukaryote RNA maturation5 end: cappingaddition of 7-methylguanosinelinked by three phosphates3 end: poly(A) tail addition of up to 200 adenine nucleotidesdownstream of AAUAAA polyadenylation signalIntron removal by spliceosomeAlmost all introns have 5GU and 3AG recognition sequence (GU AG rule)snRNPs of spliceosome provide catalysis for reactionintron excised as lariat, then destroyed

The mRNA 5-cap structureFunctions:

marks the 5-end of the first exon and aids in the splicing process

essential for nucleo-cytoplasmic transport of mRNAs through interaction with nuclear cap-binding proteins

increases the efficiency of translation by targeting formation of the preinitiation complex (cytoplasmic cap-binding proteins)

protects the transcript from 53 exoribonucleolytic activitiesNelson & Cox, 2005, p. 1008

Enzymatic reactions required for mRNA 5-cappingNelson & Cox, 2005, p. 1008

20-40 nucleotides apartSequence determinants of 3 mRNA processing3 processing of mRNA in eukaryotes(1)An enzyme complex recognizes the polyadenylation signal (AAUAAA) and a less well conserved G-U rich sequence located 20-40 nucleotides downstream.

(2)An endonuclease cleaves the primary transcript 10-30 nucleotides downstream of the AAUAAA signal.

(3)A series of 80-250 A residues are added to the 3-end of the cleaved transcript by polyadenylate polymerase.

Nelson & Cox, 2005, p. 1013

Intron splice sites and branch pointintronThree different splicing mechanisms have been identifiedGroup I intron splicingGroup II intron splicingSpliceosome

All three cases involve Removal of the intron RNA Linkage of the exon RNA by a phosphodiester bond

Splicing Splicing among group I and II introns is termed self-splicingSplicing does not require the aid of enzymesInstead the RNA itself functions as its own ribozyme

Group I and II differ in the way that the intron is removed and the exons reconnectedRefer to Figure 12.18

Group I and II self-splicing can occur in vitro without the additional proteinsHowever, in vivo, proteins known as maturases often enhance the rate of splicing

Figure 12.18

In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNAAlso as heterogeneous nuclear RNA (hnRNA)

This RNA is altered by splicing and other modifications, before it leaves the nucleus

Splicing in this case requires the aid of a multicomponent structure known as the spliceosome

Table 12.4 describes the occurrence of introns in genes of different species

Mechanism of intron splicing (1)

Mechanism of intron splicing (2)Two consecutive cleavage reactions (transesterifications) : 5 splice site (donor site) is cleaved and lariat formed by 5-2 phosphodiester bond to the 2-A at the branch site 3 splice site (acceptor) is cleaved through attack of free 3-OH from the cleaved donor site and joined to donor siteMechanism of nuclear splicing

adapted from Nelson & Cox, 2005, p. 1012

75

Synthesis and processing of ovalbumin mRNANelson & Cox, 2005, p. 101376

Differential RNA processing: Multiple mRNAs from a single geneNelson & Cox, 2005, p. 101477

Tissue-specific processing of the calcitonin primary transcriptNelson & Cox, 2005, p. 101578rRNA processing in eukaryotes

Nelson & Cox, 2005, p. 101679tRNA processing

Nelson & Cox, 2005, p. 101680

Maturation of a tRNA1324. Protein StructureProtein structure (1)Protein is polymer of amino acids (polypeptide)each amino acid has R group (side chain) conferring unique propertiesamino acids connected by peptide bondeach polypeptide has amino end (N-term) and carboxyl end (C-term)

Non-polar and hydrophobicPolar and hydrophilicNeutralBasicAcidChemical properties of amino acids

The Peptide BondProtein structure (2)Structuresprimary: amino acid sequencesecondary: hydrogen bonding, -helix and -sheettertiary: folding of secondary structure (3-D)quaternary: two or more tertiary structuresPrimary structure is determined by coding sequence of gene and will therefore determine the higher-order structure. But recall that 3-D structure (or quaternary structure) is the functional biological form of the protein

Multiplicity of protein functionThe huge diversity of protein structures can be formed , the diversity enables proteins(proteome) to carry out a variety of biological functionBiochemical catalysis (enzyme like RNA polymerase)Structure (cytoskeleton)Movement (cytoskeletal fibers)Transport (hemoglobin, albumin)Regulation (STAT :Signal transducer and activator of transcription , hormone, cytokine)Protection (antibody, thrombin)Storage (ferritin, gliadin)5. TranslationTranslation: basic conceptsmRNA is translated by tRNA at ribosomenucleotide sequence is read three nucleotides at a timeeach triplet is called a codoneach amino acid correspond to one or more codons64 possible codons (4 4 4) = genetic codeused by all organisms with few exceptionsGenetic code specifies 20 different amino acids and 3 stop codons 93The Genetic Code

allows for correspondence between triplets of bases in DNA and the amino acid sequence of a polypeptide (protein)

written or expressed in terms of RNA triplets as compared to DNA triplets because it is with messenger RNA that the translation process occurs Codons: Triplets of three bases in RNA that encode an amino acid. There are 64 possible codons (4 bases taken 3 at a time = 43)

Stop and start codons:Start = AUG (codes for methionine) - site where translation beginsStop = UAA, UAG and UGA - sites where translation ends

Degeneracy: Most amino acids have more than one codon. For example, glycine is encoded by GGU GGC GGA and GGG. 94

95

96

During translation, the genetic code in mRNA is read and converted into protein by means of the protein synthesizing machinery, which consists of ribosomes, tRNA, amino acids, and a number of enzymes.Codon translation: from RNA triplet to aatRNAanticodon consists of 3 nucleotidesbase pairs with codon in antiparallel fashion3 acceptor end attaches amino acidattachment catalyzed by aminoacyl-tRNA synthetasesone for each different tRNAThe wobble hypothesispermits third nucleotide of anticodon (5 end) to hydrogen bond with alternative nucleotidepermits a tRNA to translate more than one codon98

The structure of transfer RNA (tRNA)Transfer RNA (tRNA)Anticodon binds to complementary codon of mRNA

Secondary structure of a tRNA

Tertiary structure of a tRNA

Codon recognition by tRNATranslation at the ribosomeRibosomelarge subunit small subunit 3 ribosomal sitesA site (amino site), accepts incoming charged tRNAP site (polypeptide site), peptide bondE site (exit site), tRNA exits ribosomeAmino terminus synthesized first, beginning near 5 end of mRNA

Assembly of different ribosomesRibosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons.Each ribosome has a large and a small subunit.These are composed of proteins and ribosomal RNA (rRNA), the most abundant RNA in the cell.

Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.The P site holds the tRNA carrying the growing polypeptide chain.The A site carries the tRNA with the next amino acid.Discharged tRNAs leave the ribosome at the E site.

Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase.The 20 different synthetases match the 20 different amino acids.Each has active sites for only a specific tRNA and amino acid combination. The synthetase catalyzes a covalent bond between them, forming aminoacyl-tRNA or activated amino acid.

Formation of aminoacyl-tRNATranslation can be divided into three stages: initiation elongation terminationAll three phase require protein factors that aid in the translation process.Both initiation and chain elongation require energy provided by the hydrolysis of GTP.Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon.Initiation factors bring in the large subunit such that the initiator tRNA occupies the P site.

Sonenberg et al., eds., Translational Control ofGene Expression (2000), p. 46.Sequence of events leading totranslation initiationInitiation can be divided into four stepsRibosomal dissociation : dissociation of the ribosome into its 40S and 60S sub-units;Formation Preinitiation Complex : binding of a ternary complex consisting of met-tRNA i , GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex; Formation Initiation Complex : binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex; and Formation The 80S Initiation Complex combination of the 43S initiation complex with the 60S ribosomal subunit to form the 80S initiation complex.Initiation Step 1. Ribosomal DissociationTwo initiation factors, eIF-3 and eIF-1A, bind to the newly dissociated 40S ribosomal subunit. This delays its reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40Ssubunit.

Initiation Step 2. Formation Preinitiation Complex The binding of GTP by eIF-2 (binary complex) then binds to met-tRNAi (initiation codon AUG).This ternary complex (met-tRNAi-GTP-eIF-2) binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and eIF-1A.

the 43S preinitiation complexThe ternary complexThe binary complexInitiation Step 3(a). Formation Initiation ComplexThis methyl-guanosyl triphosphate cap facilitates the binding of mRNA to the 43S preinitiation complex. A cap binding protein complex, eIF-4F (4F), which consists of eIF-4E and the eIF-4G (4G)-eIF4A (4A) complex, binds to the cap through the 4E protein.

the 43S preinitiation complexInitiation Step 3(b). Formation Initiation ComplexThen eIF-4A (4A) and eIF-4B (4B) bind and reduce the complex secondary structure of the5 end of the mRNA through ATPase and ATP-dependent helicase activities.

Initiation Step 3(c). Formation Initiation ComplexThe association of mRNA with the 43S preinitiation complex to form the 48S initiation complex requires ATP hydrolysis.eIF-3 is a key protein because it binds with high affinity to the 4G component of 4F, and it links this complex to the 40S ribosomal subunit.

the 48S initiation complexInitiation Step 4. Formation The 80S Initiation Complex The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydrolysis of the GTP bound to eIF-2 by eIF-5. This reaction results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the 80S ribosome. At this point, the met-tRNAi is on the P site of the ribosome, ready for the elongation cycle to commence.

the 80S ribosomeElongation consists of a series of three-step cycles as each amino acid is added to the proceeding one.

STEP 1 : BINDING OF AMINOACYL-tRNA TO THE A SITEThe binding of the proper aminoacyl tRNA in the A site requires proper codon recognition.Elongation factor EF1A forms a ternary complex with GTP and the entering aminoacyl-tRNA.This complex then allows the aminoacyl-tRNA to enterthe A site with the release of EF1AGDP and phosphate.GTP hydrolysis is catalyzed by an active site on the ribosome, EF1A-GDP.Then recycles to EF1A-GTP with the aid of other soluble protein factors and GTP.STEP 2 : PEPTIDE BOND FORMATIONThe -amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P siteThis reaction is catalyzed by a peptidyltransferase, a component of the 28S RNA of the 60S ribosomal subunit.The reaction results in attachment of the growing peptide chain to the tRNA in the A site.

STEP 3 : TRANSLOCATIONThe now deacylated tRNA is attached by its anticodon to the P site at one end and by the open CCA tail to an exit (E) site on the large ribosomal subunit.At this point, elongation factor 2 (EF2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EF1A-GTP and another cycle of elongation.

The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed.

127

128

Termination occurs when one of the three stop codons reaches the A site.A release factor (eRF= RF1). RF1 is bound by a complex consisting of releasing factor RF3 with bound GTP. This complex, with the peptidyl transferase, promotes hydrolysis of the bond between the peptide and the tRNA occupying the P site.This frees the polypeptide and the translation complex disassembles.

TerminationThus, a water molecule rather than an amino acid is added.This hydrolysis releases the protein and the tRNA from the P site. Upon hydrolysis and release, the 80S ribosome dissociates into its 40S and 60S subunits, which are then recycled. The mRNA is then released from the ribosome, which dissociates into its component 40S and 60S subunits, and another cycle can be repeated.

Typically a single mRNA is used to make many copies of a polypeptide simultaneously.Multiple ribosomes, polyribosomes, may trail along the same mRNA.A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide.

During and after synthesis, a polypeptide coils and folds to its three-dimensional shape spontaneously. The primary structure, the order of amino acids, determines the secondary and tertiary structure.Chaperone proteins may aid correct folding.In addition, proteins may require posttranslational modifications before doing their particular job.This may require additions like sugars, lipids, or phosphate groups to amino acids.Enzymes may remove some amino acids or cleave whole polypeptide chains.Two or more polypeptides may join to form a protein.136

Overview of Translation137

Role of ribosomal RNA in protein synthesis

Ribosomal RNA plays a key role at all steps of protein synthesis, in recognition of initiation sequences, through rRNA-mRNA interactions during elongation, through stabilization of tRNAs, and in a catalytic way during peptide bond formation and the termination reactions.

138

Effects of antibiotics on protein synthesis

A large number of antibiotics inhibit protein synthesis in bacteria by interacting with the ribosome. These reactions are quite specific and many have been shown to interact with rRNA. Several of these antibiotics are clinically useful, including streptomycin, which inhibits initiation, and chloramphenicol and tetracycline, which inhibit chain elongation.6. Protein Function and effect of mutationsProtein functionFunction determined by amino acid sequenceColinearity between DNA nucleotide sequence and amino acid sequence of proteinTwo broad types of proteinstructural proteinsactive proteins, including enzymesProteins often have specialized functional regions called domains Proteins may consist of a single or multiple functional domains Point mutations can affect protein structure and functionMutations are changes in the genetic material of a cell (or virus).These include large-scale mutations in which long segments of DNA are affected (for example, translocations, duplications, and inversions).A chemical change in just one base pair of a gene causes a point mutation.If these occur in gametes or cells producing gametes, they may be transmitted to future generations.Malfunctioning allelesMutation alters gene function by altering structure/function in productwild-type: normal allele designated by plus (+) signexample: arg-3+mutation: change in nucleotide sequencesometimes designated by minus () sign

Types of mutationMutant site: location of nucleotide changeThree main types of mutation in coding sequence: substitution changing an amino acid (aka. nonsynonymous or nonsilent substitution) e.g., 5GGA3 5GAA3, gly gluSubstitution introducing a premature stop codone.g, 5GGA3 5UGA3, gly stopInsertion or deletion introducing a frameshift in the reading frameAny insertion or deletion of one or two nucleotides (or any length that is not a multiple of 3) result in a frame shift in the reading frame Provokes an alteration of all downstream codons Results in a truncated protein with a premature stop codon or with a different C-terminal region

For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.

A point mutation that results in replacement of a pair of complementary nucleotides with another nucleotide pair is called a base-pair substitution.Some base-pair substitutions have little or no impact on protein function.In silent mutations or samesense mutations, alterations of nucleotides still indicate the same amino acids because of redundancy in the genetic code.Other changes lead to switches from one amino acid to another with similar properties.Still other mutations may occur in a region where the exact amino acid sequence is not essential for function.Other base-pair substitutions cause a readily detectable change in a protein.These are usually detrimental but can occasionally lead to an improved protein or one with novel capabilities.Changes in amino acids at crucial sites, especially active sites, are likely to impact function. Missense mutations are those that still code for an amino acid but change the indicated amino acid.Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.

Insertions and deletions are additions or losses of nucleotide pairs in a gene.These have a disastrous effect on the resulting protein more often than substitutions do.Unless these mutations occur in multiples of three, they cause a frameshift mutation.All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons.The result will be extensive missense, ending sooner or later in nonsense - premature termination.

Mutations can occur in a number of ways.Errors can occur during DNA replication, DNA repair, or DNA recombination.These can lead to base-pair substitutions, insertions, or deletions, as well as mutations affecting longer stretches of DNA.These are called spontaneous mutations.

Mutagens are chemical or physical agents that interact with DNA to cause mutations.Physical agents include high-energy radiation like X-rays and ultraviolet light.Chemical mutagens may operate in several ways.Some chemicals are base analogues that may be substituted into DNA, but that pair incorrectly during DNA replication.Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix. Still others cause chemical changes in bases that change their pairing properties.Researchers have developed various methods to test the mutagenic activity of different chemicals.These tests are often used as a preliminary screen of chemicals to identify those that may cause cancer.This make sense because most carcinogens are mutagenic and most mutagens are carcinogenic. Effect of mutationIs extremely variable: leaky mutation: reduced protein functionnull mutation: complete loss of protein functionsilent mutation: no change in protein function, though amino acid sequence may be changedMutations may also occur in non-coding sequence, affecting information transfer: mutations in exon-intron junction, may affect splicingmutations in promoter or regulatory sequences, may affect the level of expression of a protein mutations in UTRs, may affect level or timing of expression of a protein

156 THANK YOU FOR YOUR ATTENTION