Chapter 16: ReviewChapter 16: ReviewMolecular Basis of Molecular Basis of InheritanceInheritance

•Search for genetic material led to DNA

•Discovery- DNA double helix•DNA replication: Basics•DNA repair

Chapter 17: Chapter 17:

From Gene to From Gene to ProteinProtein• Study of metabolic defects

provided evidence that genes specify proteins

• Transcription & translation are main processes linking gene to protein.

• Genetic code: nucleotide triplets specify amino acids

• Transcription is the DNA-directed synthesis of RNA

• Signal peptides target some eukaryotic polypeptides to specific destinations in the cell

• RNA plays multiple roles in the cell• Compare protein synthesis in

prokaryotes & eukaryotes.

•A point mutation can affect the function of a protein

•Ask again, what is a gene?

Study of Metabolic Study of Metabolic Defects Provided Evidence Defects Provided Evidence that Genes Specify that Genes Specify ProteinsProteins• Archibald Garrod (1909)

proposed relationship between genes & proteins (alkaptonuria, dark urine)

• George Beadle & Edward Tatum (1930s) demonstrated the relationship between genes & enzymes while studying a bread mold with mutants.

How Do Genes How Do Genes Control Control MetabolismMetabolism• Beadle & Tatum• Red bread mold (maybe you know it)• Wild-type strain has minimal

requirements.• Multi-step pathway to synthesize the

amino acid arginine from a precursor• Three classes of mutants unable to

metabolize arginine.

Neurospora - Red Bread Mold

• They concluded that each mutant was defective in a single gene coding for one enzyme.

• This lead to the one gene-one enzyme hypothesis: the function of a gene is to dictate the production of a specific enzyme.

Modified IdeaModified Idea•This idea was modified to be one gene-one polypeptide hypothesis because: not all proteins are enzymes (keratin, insulin).

•Many proteins are made of several polypeptides (hemoglobin, 2 chains).

Transcription & Transcription & Translation:Translation: Two Main Two Main Steps from Gene to ProteinSteps from Gene to Protein•Genes are the instructions for making specific proteins, but a gene does not build a protein directly.

•The bridge between genetic information and protein synthesis is RNA, ribonucleic acid (Chap. 5).

DNA & RNA: Both DNA & RNA: Both PolymersPolymers

• Both RNA & DNA are nucleotide polymers with two main differences: deoxyribose of DNA has one less hydroxyl group than ribose (sugar); the other difference is the nitrogenous base-thymine (T) is unique to DNA & uracil (U) unique to RNA.

DNA & RNA Structural Differences

• Flow of information - gene to protein, is described in linguistic terms because both nucleic acids and proteins have specific sequences of monomers, much as specific sequences of letters communicate information in the written word.

•DNA/RNA - monomers are the four types of nucleotides (nitrogenous bases differ) that are 100’s or 1000’s of nucleotides long, each gene having a specific base sequence.

•A protein has monomers in a particular linear order, but the monomers are......?

Amino AcidsAmino Acids•Amino acids..20 of them.•Thus, nucleic acids & proteins contain information written in two different chemical languages....

•Getting from one to the other requires two major steps:

Transcription and Translation

TranscriptionTranscription

• Transcription is the synthesis of RNA under the direction of DNA (template...recall replication). Both nucleic acids use the same monomeric language, the information just has to be transcribed (copied) from one molecule to another.

•The RNA molecule made according to the DNA template is a transcript of the gene’s protein-building instructions.

•This RNA is called mRNA (messenger RNA), which functions as a genetic message from DNA to the protein-synthesizing machinery.

•Translation is the actual synthesis of a polypeptide, which occurs under the direction of mRNA.

•There is a change in language from the base sequence of an mRNA into the amino acid sequence of a polypeptide.

•Ribosomes are the sites of translation, which are made of numerous enzymes & other agents that facilitate the orderly linking of amino acids.

Prokaryote/Eukaryote Prokaryote/Eukaryote Difference?Difference?

•Bacteria lack a nuclei, so DNA is not segregated from ribosomes, etc.

•Thus, transcription & translation are coupled, with ribosomes attaching to the leading end of an mRNA molecule while transcription is still ongoing. (Fig. 17.2, CD).

Prokaryotic Cell

Immediate Translation

No Nucleus

Eukaryotic Cell

Nucleus

RNA Processing

•DNA RNA Protein

Genetic Code: Nucleotide Genetic Code: Nucleotide Triplets Specify Amino AcidsTriplets Specify Amino Acids

•Only four nucleotides code for 20 amino acids.

CodonCodon• Three nucleotide “words” are

called codons.• Codon=three-nucleotide

sequence in mRNA that specifies which amino acid will be added to a growing polypeptide or that signals termination.

• Codon is basic unit of genetic code.

Recall.......Recall.......• Genes are not directly translated

into amino acids, but are first transcribed as codons into mRNA.

• Only one strand is transcribed, the other non-template strand serves as a parental strand for making a new template when DNA replicates.

• An mRNA is complementary to the DNA template from which it is transcribed, for example, the DNA sequence CCG is the codon for glycine, the complementary mRNA transcript is GGC. Uracil substitutes for thymine & pairs with adenine (Fig. 17.3).

Triplet Code

DNA

mRNA

AminoAcid

Fig. 17.4 Triplet Fig. 17.4 Triplet code.code.• Each mRNA codon specifies which

one of the 20 amino acids will be incorporated into a corresponding position in a polypeptide.

• The number of nucleotides making up a genetic message is 3 times the number of amino acids.

Cracking the Genetic Cracking the Genetic CodeCode

•By the mid-1960s all 64 codons were known. Figure 17.4 is the dictionary of the genetic code.

GENETIC CODE

AUGMet. =Start

Stop=UAAUAGUGA

FactoidFactoid• AUG that codes for methionine (Met, start signal or initiation codon) is sometimes removed subsequently.

•There is redundancy in the code....several codons for one amino acid, but there is no ambiguity (same codon for two amino acids). Codons for the same amino acid may differ only in the third base of the triplet.

Reading Reading FrameFrame•Reading frame = correct grouping of adjacent nucleotide triplets into codons that are in the correct sequence on mRNA.… nonoverlapping three-letter words.

Reading frameReading frame• THE BIG RED CAT ATE THE

BIG BAD FAT RAT.

• HEB IGR EDC ATA TET HEB IGB ADF ATR AT-

•My codon is A D D.

–Attention

–Deficit

–Disorder•What is your codon?

The Genetic The Genetic CodeCode•The genetic code is nearly universal - a language that is shared across all of life meaning it must have been operating very early in the history of life.

Transcription Up Transcription Up CloseCloseTranscription is the DNA-Transcription is the DNA-DirectedDirectedSynthesis of RNASynthesis of RNA•RNA polymerases pry apart

the two DNA strands & hook together the RNA nucleotides as they base pair along the template beginning at the 3’ end.

•DNA that is transcribed into an RNA molecule is the TRANSCRIPTION UNIT.

Transcription Up Transcription Up CloseClose• Prokaryotes have one RNA

polymerase. Eukaryotes have three.• mRNA synthesis-RNA polymerase II•Transcription steps:

1) polymerase binding & initiation 2) elongation 3) termination

RNA Polymerase RNA Polymerase Binding & InitiationBinding & Initiation•RNA polymerases bind to DNA regions called PROMOTORS.

•Promoter = initiation site + dozens of nucleotides “upstream” from initiation site.

e.g. TATA box (eukaryotes) are 25 bases upstream.

•TRANSCRIPTION FACTORS - aid polymerases in finding promotor regions on DNA (sometimes attach before polymerase can bind).

ElongationElongation•RNA polymerase II untwists DNA one turn (helix) at a time exposing 10 bases for pairing of RNA nucleotides at the 3’ end.

•mRNA peels away as the noncoding strand reforms the double helix.

•A single gene can be transcribed simultaneously by several polymerase IIs so it can produce proteins faster (more copies of mRNA).

TerminationTermination

•RNA polymerase transcribes until termination site is reached (AAAAAA in eukaryotes).

•Play CD #2

Figure 17.6

Figure 17.7

Initiation of Transcription ata Eukaryotic Promoter

Eukaryotic Cell Modify RNA Eukaryotic Cell Modify RNA after Transcriptionafter Transcription

•Alternation of mRNA Ends•Split Genes and RNA Splicing

•Ribozymes

RNA ProcessingRNA Processing: Both ends : Both ends (5’ & 3’) are modified, then (5’ & 3’) are modified, then cut apart & spiced together cut apart & spiced together againagain•Alteration of mRNA ends - in the nucleus

- 5’ Cap protects mRNA & is an “attach here” signal for small ribosomes.

• - 3’ Poly-A tail (several hundred adenines) for protection & transport from nucleus to cytoplasm.

RNA Processing: RNA Processing: Add Cap & Add Cap &

TailTail

5’Cap 3’Tail

RNA ProcessingRNA Processing: :

•RNA splicing - Average DNA molecule is 8000 bases, RNA in nucleus is same length, but 1200 nucleotides to code for protein of 400 amino acids.

•What happened to the 6,800 nucleotides between the nucleus and cytoplasm....?

RNA ProcessingRNA Processing: :

• INTRONS - intervening noncoding segments of DNA are transcribed then removed.

•EXONS - are coding regions that are express eventually (translated into proteins).

•RNA splicing occurs for tRNA & rRNA & mRNA. (see Figure 17.9).

RNA Processing: Splicing

Transcription

Introns Excised

Exons Spliced

Play CD Play CD

Synthesis of Synthesis of ProteinProteinTranslation Up Close (p. Translation Up Close (p. 304)304)• In the Process of Translation a

cell interprets a genetic message & builds a protein.

•The message is a series of codons along the mRNA molecule

•The interpreter is another type of RNA - TRANSFER RNA (tRNA).

TRANSFER RNA TRANSFER RNA

•tRNA transfers amino acids from the cytoplasm’s amino acid pool to the ribosome.

•Cell keep the pool stocked with 20 amino acids it makes or takes up from the surrounding solution.

•Some 45 different tRNA molecules associate with particular mRNA codons that code for amino acids.

Translation Up CloseTranslation Up Close

• On one end is a tRNA’s particular amino acid, on the other end is a base triplet called an ANTICODON

• The anticodon binds according to the base-pairing rules to a mRNA codon.

• Ribosomal enzymes join amino acids into a chain.

Transfer RNATransfer RNA• tRNAs are transcribed in the nucleus from DNA templates (nucleus) then travel to cytoplasm for translation where they are used repeatedly.

•Some tRNAs have anticodons that recognize two or more codons. The third base pairings (between anticodon & mRNA) are not as strict.

•This relaxation of base-pairing rules is called WOBBLE.

Explains synonymous codons for certain amino acids.

•See Figure 17.13 for aminoacyl-tRNA synthase joining a tRNA to an amino acid......

RibosomesRibosomes•Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis.

•Ribosomal unit = proteins (large & small subunit) + ribosomal RNA (rRNA)

Building a Building a PolypeptidePolypeptide

• Initiation>Elongation> Termination

Building a Building a PolypeptidePolypeptide

•Initiation : mRNA + tRNA (with amino acid) + two ribosomal subunits + GTP (energy).

Initiation of Translation

Building a Building a PolypeptidePolypeptide

•Elongation: Codon recognition (+GTP) > Peptide bond formation (peptidyl transferase) & release of amino acid from tRNA

Elongation of TranslationElongation of Translation

Codon RecognitionCodon Recognition

Peptide Bond FormationPeptide Bond Formation

Building a Building a PolypeptidePolypeptide

•Translocation: tRNA dissociates from ribosome....mRNA & ribosome move in unison (ratchet-like).

Elongation of TranslationElongation of Translation

TranslocationTranslocation

A A SiteSite

P P SiteSite

Building a Building a PolypeptidePolypeptide• Termination: Elongation until

TERMINATION CODON reached (UAA, UAG, UGA - not amino acid codons)Release factor hydrolyzes the completed polypeptide from the tRNA, freeing the polypeptide from the ribosome.

• See Figures 17.14 - 17

Termination of TranslationTermination of Translation

Free PolypeptideFree Polypeptide

TerminationTerminationCodonCodon

Ribosome Ribosome DissociationDissociation

Play CDPlay CD

Building a Building a PolypeptidePolypeptide•POLYRIBOSOMES - more than one ribosome translating on same mRNA molecule

(Figure 17.18 in book).

Signal PeptidesSignal Peptides•Free (cytosol) & bound (to endoplasmic reticulum) ribosomes - Free - proteins in cytosolBound - membrane proteins & proteins to be secreted.

•SIGNAL SEQUENCE - on peptide enables ribosome to attach to receptor site on ER membrane (signal seq. is eventually removed).

Signal Mechanism for Targeting Proteins

SignalRecognition Particle

SignalSequence

ReceptorSite

Endoplasmic Reticulum

Types of RNA in a Types of RNA in a EukaryoteEukaryoteSee Table 17.1See Table 17.1•Messenger RNA•Transfer RNA•Ribosomal RNA•Primary transcript•Small nuclear RNA•Signal recognition particle

MutationsMutations•MUTATIONS are changes in the genetic makeup of a cell.

•POINT MUTATIONS - chemical changes in just one nucleotide in a single gene. e.g. gene in gamete with point mutation may be passed on to next generation (genetic disorder)

Types of MutationsTypes of Mutations

•SUBSTITUTIONS: base-pair is replaced by another pair of nucleotides. Net result is a MISSENSE mutation that still codes for an amino acid.

•May not be a problem unless it is a termination codon (NONSENSE MUTATION).

Types of MutationsTypes of Mutations

• INSERTIONS & DELETIONS: addition or loss of a nucleotide pairs in a gene. This more disasterous as may throw off reading frame causing a FRAMESHIFT MUTATION.

•Unless three nucleotides are added or deleted, or the mutation is near the end of the gene, the protein is likely to be nonfunctional.

Molecular Basis of Sickle-Molecular Basis of Sickle-Cell Disease: A Point Cell Disease: A Point MutationMutation

Categories & Consequences Categories & Consequences of Point Mutations (Fig. of Point Mutations (Fig. 17.22)17.22)

MutagensMutagens•MUTAGENS - physical or chemical agents that interact with DNA to cause mutations (e.g. x-rays, UV light).

•AMES TEST - for mutagenic strength of chemicals (pesticides, drugs for mutagenic & cancer-causing potential) see text.

See Figure 17.23See Figure 17.23Transcription & Transcription & TranslationTranslation