Genetics Exam 3

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Transcription

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Conversion of DNA to RNA
Nucleotide to Nucleotide

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Translation

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Conversion of RNA to protein
Nucleotide to amino acid
Polymerization of amino acids into polypeptide chains

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Reading Frame

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the sequence of codons that runs from a specific start codon to a stop codon

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mRNA and reading frames

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mRNA can be translated in any one of 3 reading frames
depends on where translation begins
only 1 of the 3 encodes for the protein
Translation start signal- determines where translation begins (AUG)
*formylmethionine in prokaryotes
methionine in eukaryotes

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Adaptor Hypothesis (Translation)

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proposed in 1957 by Francis Crick
transfer RNA (tRNA) adapts each codon triplet in mRNA to the correct amino acid
Anticodon in a tRNA pairs with a codon in mRNA

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tRNAs (translation)

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75-90 nt long, transcribed as a larger precursor molecule (by RNA Pol III in eukaryotes)
Processing is required for the tRNA to fold properly
About 10% of the nt in a tRNA molecule are modified forms of G,U,C or A ribonucleotides
*these are important for tRNA conformation and base pairing of the anticodon, especially pairing at the wobble site

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structure of tRNA

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TCG loop, acceptor stem, D loop, variable loop, anticodon loop

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Redundancy of the Genetic Code- the wobble postion:

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61 codons for 20 amino acids
more than one tRNA for each amino acid
the same tRNA can base pair with more than one codon
both occur: multiple tRNA for many AAs, Wobble position (mismatches at 3rd position can be tolerated)

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Wobble position

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many alternate codons differ only at the 3rd nt
Bacteria: 31 tRNAs for the 61 codons
Eukaryotes: 497 tRNA genes (only 48 different tRNA codons)

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Charging tRNA molecules

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formation of the covalent link b/w a tRNA molecule and a correct amino acid
Aminoacyl-tRNA synthesis

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Aminoacyl-tRNA synthetase

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20 different enzymes (one for each amino acid)
Covalently couples the amino acid to its appropriate set of tRNA molecules
*e.g. one enzyme attaches glycine to all tRNAs that recognize codons for glycine
coupling reaction is ATP dependent

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Aminoacyl-tRNA synthetase: coupling reaction

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ATP-dependent
results in the formation of a high energy bond between the amino acid and the tRNA
used for formation of peptide bonds during protein synthesis

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Decoding process for Translation of Nucleic Acid Sequences into Amino Acid Sequences

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1. Aminoacyl tRNA synthetase couples the specific amino acid to the appropriate tRNA through a high energy ester bond
2. Three base sequence in the tRNA (anticodon) base pairs with the codon in the mRNA specifying the attached amino acid

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Ribosomes- the site of Protein Synthesis

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Ribosomes are composed of about 50 different proteins (ribosomal proteins) and several RNA molecules (rRNA) *Bacteria ~1000 ribosomes, Eukaryotic cells ~ several million ribosomes

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Prokaryotic and Eukaryotic ribosomes are similar in design and function

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large and small subunit form a complete ribosome
small subunit provides the framework for matching tRNAs with the correct codons in the mRNA
large subunit provides the catalytic function- forms the peptide bonds linking AAs into a polypeptide chain

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Prokaryotic Ribosomes v. Eukaryotic

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Prokaryotic: 50s + 30s; 31 + 21 proteins
Eukaryotic: 60s + 40s; 49 + 33 proteins

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Translation: 3 steps

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Initiation, elongation, termination

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Translation- Initiation- E. coli

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Initiation in E. coli: mRNA associates with small ribosomal subunit
Charged tRNA, GTP, Mg++, and translation initiation factors (IF proteins)
Bacteria-Initiator tRNA encodes for fmet

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Translation-Initiation- Shine-Delgarno Sequence

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6 ribonucleotide sequence element found preceding the translation start codon
Contains only purines and base pairs with a region found in the 16s rRNA of the small subunit

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Translation components (Initiation)

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Small/Large subunit
GTP
Triplet codons
Anticodon (initiator tRNA) w/fmet
Elongation factors
Initiation factors

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Translation Initiation: Prokaryotes: 3 steps

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mRNA binds to small subunit along w/initiation factors
2. Initiator tRNA(fmet) binds to mRNA codon in P site, IF3 released
3. Large subunit binds to complex, IF1 and IF2 released; EF-Tu binds to tRNA, facilitating entry into A site

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Translation-Elongation

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Once the two subunits come together, 2 binding sites for charged tRNAs are formed: P (peptidyl) site and A (aminoacyl) site

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Peptidyl transferase

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catalyzes formation of the peptide bond b/w two amino acids: takes place while the AA is bound to the tRNA

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Peptide bond

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covalent bond b/w two AAs

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Translation Elongation in Prokaryotes

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1. Second charged tRNA enters A site, facilitated by EF-Tu
2. Peptide bond forms, tRNA moves to the E site and out of the ribosome, mRNA has been translocated three bases to the left, resulting in the tRNA bearing the dipeptide to shift into the P site

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Translation Elongation in Prokaryotes: steps 3 & 4

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3. first elongation step complete, facilitated by EF-G. 3rd charged tRNA ready to enter the A site
4. 3rd tRNA has entered A site, facilitated by EF-Tu; second elongation step begins

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Translation Elongation: steps 5, 6

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5. Tripeptide formed; second elongation step completed; uncharged tRNA moves to E site
6. Polypeptide chain synthesized and exiting ribosome

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Translation- Termination

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Signaled when the ribosome encounters a "stop codon" in the A site (UAG,UAA,UGA)
Release factors (RFs) cleave the polypeptide chain from the last tRNA: dependent on hydrolysis of GTP, Polypeptide is released through a pore in the large ribosomal subunit
tRNA moves to the E site and the polypeptide is released

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Polyribosomes and Signal Amplification

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Multiple ribosomes can simultaneously translate the same MRNA molecule
As soon as the 5' end of an mRNA emerges from the ribosome, translation can be initiated by a new ribosome
Signal amplification- a single gene is transcribed into thousands of mRNA molecules, each of which gives rise to thousands of polypeptides

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polycistronic

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of or pertaining to the transcription of two or more adjacent cistrons into a single messenger RNA molecule

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Translation in Eukaryotes

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5' mRNA cap- 7 (methylguanosine)
Kozak Sequence
Initiation-Elongation-Termination factors also important in eukaryotic cells
Ribosomes are cytoplasmic and associated w/the ER

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Kozak Sequence

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Nucleotide sequence surrounding the translation start codon (AUG)
- 5' ACCAUG 3' : The C and G residues most important
- Functions similar to the Shine- Delgarno sequence in bacteria

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Protein Synthesis Inhibitors- Useful as Antibiotics: only in Bacteria

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Tetracycline: blocks binding of tRNA to A site
Streptomycin: prevents transition from initiation complex
Chloramphenical: blocks peptidyl transferase rxn on ribosomes
Erythromycin: blocks translocation rxn on the ribosome
Rifamycin: inhibits RNA synthesis

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Antibiotics: Protein Synthesis inhibitors in Bacteria and Eukaryotes

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Puromycin: causes premature release of polypeptide chain
Actinomycin D: binds to DNA and blocks RNA Pol II

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Protein Synthesis Inhibitors only in Eukaryotes:

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Cycloheximide: blocks translocation rxn on the ribosome
Anisomycin: blocks peptidyltransferase rxn on ribosomes

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Defects in Metabolic Pathways

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Archibold Garrod: Studied inherited metabolic diseases in humans
Caused by faulty "ferments" (enzymes) and are the result of "inborn errors of metabolism"
Metabolic Pathways: Starting product, one or several intermediates, an end product

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Connecting Hereditary Changes w/Metabolic Diseases: Alkaptonuria

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Diseases in Phenylalanine and Tyrosine metabolic pathways:
Alkaptonuria: Individuals cannot oxidize homogentisic acid: results in accumulation of homogentisic acid in cells and tissues
Increased dietary protein or adding phenylalanine or tyrosine to the diet causes increase in homogentisic acid in urine of affected individuals
Autosomal, recessive disease
*Inherited conditions are due to the lack of a critical substance?*

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Phenylketonuria (PKU)

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Defect in some metabolic pathway as alkaptonuria
Autosomal Recessive disorder- defective enzyme is pehnylalanine hydroxylase
Accumulation of phenylalanine and phenylpyruvic acid: enter the brain and cause mental retardation
Studies on alkaptonuria and PKU demonstrated that many human diseases have a genetic basis

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One-gene: One- enzyme Hypothesis

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Beadle and Ephrussi: First to link biochemical errors to the loss of enzyme function
Beadle and Tatum: Induced nutritional auxotrophic mutations in Neurospora: Identified defects in specific metabolic pathway by Nutritional mutations cause the loss of essential enzymatic activity
Mutations potentially could be identified for nearly all enzymatic reactions

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Genetics to study Biochemical Pathways: Srb and Horowitz

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Studied arginine synthesis in Neurospora
Identified 7 mutant strains that required arginine for growth
Supplemented media: 2 precursors of arginine (citrulline or ornithine)
-arginine
Growth of mutant strains: Four mutant strains could grow on media supplemented w/citrulline or ornithine as well as arginine
Two mutant strains could grow on media supplemented with citrulline or arginine
One mutant strain could grow only on media supplemented with arginine.

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One-Gene; One-Polypeptide

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Not all proteins are enzymes- one gene: one protein is more appropriate
Some proteins have multiple subunits: one gene: one polypeptide is most appropriate

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Sickle Cell Anemia

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Provided first evidence that genes specify proteins other than enzymes
Erythrocytes (RBCs) become elongated under low oxygen tension- causes aggregation of RBC on venous side of the capillaries
Neel and Bea (1949) showed the disease is inherited as a Mendelian trait and is due to a defect in a single gene

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Sickle-Cell Anemia & Linus Pauling

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Linus Pauling & Vernon Ingram: determined that the defect in Sickle-Cell anemia is in the beta chain of adult hemaglobin
Defect caused by a point mutation: substitution of a valine residue for a glutamic acid at the 6th position of the beta chain
Demonstrated the molecular basis for Sickle-Cell Anemia: also demonstrated that a single gene provides the genetic information for a single polypeptide chain

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Protein Structure and Function

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Nucleotide sequence determines amino acid sequence (primary structure)
The AA sequence determines the secondary structure: localized folding of parts of the polypeptide chain: e.g. alpha sheets
Polypeptides further folded into 3' structure: overall conformation of the polypeptide chain
Many polypeptides function as complexes (tertiary structure)
Structure determines function: any mutation that affects a proteins structure will affect its function

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Basic AA structure

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Different R group in each aa divided into 4 main classes based on characteristics of R group
Proteins always have a free amino terminus at one end (N-term) and a free carboxyl group at the other end (C-term): Co-linearity of DNA, RNA, and proteins

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Posttranslational MOdificaitons of Proteins: Alteration of the N-terminal and C-terminal AAs

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bacteria- formyl group is removed from formylmethionine
Eukaryotes- methionine is either removed or modified

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Posttrans Mods of Proteins: Modificaiton of individual AAs

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Ser/Thr and Tyr can be phosphorylated by kinases
Lys can be acetylated by acetylases
Lys, Arg, His can be methylated methylases
These mods (especially phosphorylation) are important in regulating the activity/fxn of proteins

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Posttrans Mods of Proteins: Glycosylation-Addition of CHO side chains

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Glycoproteins- usually found on the external surface of the cell
ABO blood group is one example

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Posttrans Mods of Proteins: Removal of Signal Sequences

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Signal sequences- direct proteins to their proper subcellular location
e.g. membrane bound or secreted proteins have signal sequence that directs them to the ER during protein synthesis

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Posttrans Mods of Proteins: Association of metal ions with polypeptide chains

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Hemoglobin contains four iron atoms, one in each of the four polypeptide chains
Many proteins also contain zinc