Chapter 17DNA Expression

TranscriptionTranslation

Central Dogma –now we know it’s not totally correct since not all RNA is translated into protein

DNA RNA ProteinDNA DNA via replicationDNA RNA via transcriptionRNA protein via translation

Beadle and Tatum’s Theory:One gene One protein

Which is a correct statement of central dogma?A. ProteinRNADNAB. RNAproteinDNAC. DNARNAprotein

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Today we know that really one gene one RNA, but RNA is not always translated to protein

Which type of RNA is correctly paired with its function?A. rRNA: ribozymes in ribosome which

catalyze production of peptide bondsB. tRNA: code the order of nucleotides

in a polypeptideC. mRNA: deliver amino acids into the

ribosomeD. snRNA: prevent expression of a

gene, either by targeting its RNA for destruction Or by forming a double stranded DNA that can not be translated.

E. siRNA & miRNA: part of a complex that attaches to the junctions of exons and introns during alternative splicing

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Central Dogmatranscription

translation

transcription

RNA processing

translation

Which is NOT a difference in gene expression for eukaryotes and prokaryotes?

A. Prokaryotes do not splice out exons prior to translation, but eukaryotes do.

B. Prokaryote mRNA is generated in the cytoplasm & also translated in the cytoplasm

C. Eukaryotes can produce several different proteins from the same gene, according to how its processed, but prokaryotes can not.

D. Prokaryotes use a simpler genetic code than eukaryotes.

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What is the evidence that genes code proteins? Beadle & Tatum: One gene one enzyme (protein)

Yeasts able to produce all 20 amino acids via metabolic pathways (several enzymes working sequentially) were treated with radiation to create mutations. They cloned mutated cells, then tested them one by one to find which could live only with amino acid arginine added to the medium (or one of the 2 precursor products in the 3 step metabolic pathway).

They concluded that one gene codes for one enzyme; now we generalize to one gene1 protein

Events of gene expression (in eukaryotes, each gene is transcribed independently of each other gene.

Antisense strand contains promoter

sequence where RNA polymerase binds

5’ methyl cap added, in some polyA tail added, alternative splicing by snRPs

Triplet Code—mRNA codons: sequence of 3 ribonucleotides that control insertion of one amino acid

The genetic code is degenerate (the 3rd base seems not as important for

defining the amino acid coded by a codon—wobble hypothesis).

It is also redundant: 64 codons for only 20 amino acids.The

Note Note the the methionine codon, AUG, is the start codon, but it also

appears later in the sequence.

Stop codons are bound by tRNA’s which don’t get charged with an amino acid;

these are termed “release factors” because the ribosome releases the

mRNA & polypeptide at a stop codon.

RNA polymerase binds at the promoter sequence of the gene, then reads the 3’to 5’ oriented strand & produces a single stranded mRNA in the 5’ to 3’ direction.

The RNA polymerase may add a polyA tail code if this sequence is coded by the gene. The RNA polymerase disassociates from the gene at the termination site

sequence.

Open reading frame—from promoter to terminaor

How is transcription different from replication?

Replication copies all of the genome (every nucleotide—all 3 to 5 billion in each 23 chromosomes of the human cell (so 6 to 10 billion in each cell)

Transcription only copies one side (template strand) of one gene in the genome

most genes are present in one copy

Uracil instead of Thymine in RNA

3’ C or ribonucleotides has an –OH Functional group, but in deoxyribonu-Cleotides does not.

Prokaryotes can have translation happening at the same time as transcription—no nucleus, no introns

Eukaryotes must remove introns between transcription and translation, as well as adding a3’ poly A tail (controls longevity) and a 5’ guanine cap (essential for ribosome assembly) (prok do have methyl cap & some a poly A tail)

Processed DNA is sent to the cytoplasm where ribosome complexes assemble at the start codon (fmet), then translation occurs.

Bacterial translation is rapid, beginning as soon as the RNA begins separation from the DNA. Polyribosomes (several traveling on same RNA) amplify gene expression.

In both prokaryotes and eukaryotes, RNA is destroyed when protein synthesis is no longer needed, by RNAase enzymes.

Allowing DNA to avoid destruction by RNAases are several shape differences between DNA & RNA, such

that RNA is a substrate for RNAase, but DNA is not.T in DNA vs U in RNADouble stranded DNA vs single stranded RNA2’ C bonded to 2 H in DNA but to an H and an -OH in RNA

Double stranded RNA does occur is found when an RNA retrovirus infects a host, and cells have evolved to destroy ds RNA with enzymes; this is why miRNA & siRNA can downregulate the expression of a gene during normal development, too.

.

Once processed, the triplet code (genetic code) is used to control the order of amino acids in the protein

The triplet code is the order of ribonucleotides that codes one amino acid—note stop, start, “wobble” (often, the 3’ nucleotide differs in codons coding for the same amino acid.

Sense strand is read 3’ to 5’, starting at a particular distance from the promoter TATAAA box

Transcription factors bind the 5’ untranscribed region of the gene (5’ UTR), making the promoter sequence accessible to RNA polymerase, allowing initiation of transcription. Then RNA polymerase adds ribonucleotides (5’3’)complementary to the sense (template strand) of the gene. At the termination signal, the RNA polymerase detaches, then the new RNA is processed before translation.

Pre-RNA processing in the nucleus: Spliceosomes remove introns at pallindromic sequences between introns. Alternative splicing allows different exons to be used to make different versions of proteins coded by the same gene.

Signals received by cells (signal molecules like hormones and receptor proteins) convey info about how to splice the RNA, allowing alternative splicing.

Once RNA is processed, ribosomes translate it “in frame” Initiation: Ribosome complex

formation: large subunit P site attached to f-met tRNA complementary base paired to the start codon, AUG, & to a small ribosomal subunit

Elongation: Ribosome translocates (GTP energy) 5’ to 3’ one codon at a time—new tRNA to A site, then to P site (where it transfers its amino acid & growing peptide chain to the amino acid in A site, then out the E site, until termination when it reaches a stop codon where a release factor binds the stop codons UGA, UAG, UAA.

tRNA decodes the mRNA by having its anti-codon bind to a complementary codon in the A site of the ribosome.

Aminoacyl transferase enzyme loads up the amino acid—the anticodon fits into the active site at the

dame time as the correct amino acid

After translation, proteins are modified (post translational modification) and transported. Signal peptides (several amino acids at the amino terminus of the protein) define the target.

Types of post-translational modifications:

Trimming off signal peptides once the peptide has been transported into the RER.

Adding sugar chains (glycoproteins) in the RER or golgi or lipid chains (lipoproteins) in the RER—allows interaction with other molecules or attachment to membranes.

e.g., cell identification molecules that extend into the extracellular matrix are often glycoproteins

Phosphorylation—alter shape and activity or interaction with other proteins

Chapter 17 in-class essay writing quiz—one of the following essays was chosen at random 10/27/09.

(scored out of 10 points, % of 20 pts awarded)1. Describe steps that occur during transcription of a

gene. Identify molecules involved, and describe the functions of each. Include initiation, elongation, and termination. (purple choice)

2. Describe steps of post-transcriptional regulation. List the events, and describe the function (importance) of each modification. (red choice)

3. List & describe steps of translation. Identify the molecules and organelles involved. (teal choice)

Mutations

Mutations that affect the coding sequence of a protein fall into several categories:

Point mutations: only one nucleotide changedmissense—substitute one amino acidnonsense—create early stop codon

Insertion—add nucleotidesDeletion—remove nucleotidesFrameshift—add or remove multiples not equal to

3 (change entire remaining reading frame)Translocation—move entire section of gene

somewhere else in the genomeInversion—turn a section of gene backwards

Mutations can occur at the level of DNA or RNA.

RNA isn’t proof read, so mistakes occur more often in RNA.

Since many copies in RNA are made in each round of transcription, then a mistake in RNA rarely has a significant impact on the phenotype.

Mutations can occur in the codons of a gene (the coding region) or in the untranscribed/translated regions (5’ & 3’ UTRs) of a gene where regulatory proteins bind.

When do mutations affect phenotype?

When the gene is expressed (transcribed then translated) in the wrong cells or in abnormal amounts or atabnormal times

These mutations occur in the 5’ UTR (untranslated region) and the 3’ UTR, outside of the coding region of the gene. They alter transcription factor binding.

When do mutations affect phenotype? Mutations may change the phenotype if post-transcriptional exon splicing is altered:

i causing different proportions of the possible variants of the protein to be made by alternative splicingOR ii causing errors in splicing—e.g., creating frameshift s by inserting or deleting nucleotides near palindromes at the exon/intron boundaries.

This type of mutation may occur outside of the coding region of the gene, in the UTRs or inside the introns.

A mutation affects phenotype if it changes the size or shape of a protein so that either its longevity, interaction with

regulators, or ability to function is altered

These mutations occur in coding region (exons) & affect phenotype IF the mutation changes one or more amino acids.

Some amino acid substitutions (missense) have greater impact

than others.Conservative missense mutations substitute an

amino acid with similar properties, such that 3D protein shape is altered very little.

e.g., if a nonpolar amino acid is substituted for another nonpolar amino acid of similar size

Silent mutations change a codon to a replicate codon for the same amino acid.

Mutations often affect phenotype when a stop codon is inserted at least several amino acids prior to the normal stop codon since the protein is likely to fold

incorrectly after such a nonsense mutation.

Mutations that create frame shifts in the coding region often affect phenotype since changing every 3’ codon after the mutation likely alters protein shape.

Use 30 minutes to write an answer. Due 10/29/09.A List the types of mutations that change the coding

region of a gene. Describe how each type of mutation alters the DNA,the RNA, & the protein.

B Explain why mutations in the untranscribed & untranslated regions of genes can affect phenotype.

Differential expression of DNA Lab 10/28/09 Hypothesis: Since peroxidase enzyme (catalyzes breakdown of peroxide) is abundant in detoxifying cells, then we expect higher peroxidase enzyme gene expression in liver than muscle.Compare the content of peroxidase enzyme in 1 g of macerated

(mashed) chicken liver to the amount in 1 g macerated chicken leg muscle.

Add the tissue extract to 10 ml 1.5% peroxide, then swirl and wait 1 minute. Add 10 ml 1 M H2SO4 to denature the enzyme. Remove 5 ml of the mixture and titrate to 30s color change with 2% KMnO4 . Record the start and stop volumes, and the volume change.

Whichever uses LESS KMnO4 contains LESS of the enzyme since the reaction of KMnO4 with undegraded peroxide is clear—once all the undegraded peroxide is reacted, excess purple permanganate ions make the solution purple. Less enzyme will leave more peroxide needing reacted.