e-Learning Session

15.1. The Function of Genes

A. Investigators Recognize Gene Activity

1. English physician Sir Archibald Garrod introduced phrase inborn error of metabolism.
1. Garrod said inherited defects could be caused by the lack of a particular enzyme.
2. Knowing that enzymes are proteins, Garrod suggested link between genes and proteins.

B. Genes Specify Enzymes

1. George Beadle and Edward Tatum X-rayed spores of red bread mold, Neurospora crassa.
2. Discovered some resulting cultures lacked a particular enzyme for growth on medium.
3. They found that a single gene was mutated, which resulted in the lack of a single enzyme.
4. They stated one gene-one enzyme hypothesis: one gene specifies synthesis of one enzyme.

C. Genes specify a Polypeptide

1. Pauling and Itano compared hemoglobin in red blood cells of persons and normal individuals.
2. Discovered chemical properties of chain of sickle-cell hemoglobin differed from normal hemoglobin by using electrophoresis to separate molecules by weight and charge.
3. Pauling and Itano formulated the one gene-one polypeptide hypothesis: each gene specifies on poly peptide of a protein, a molecule that may contain one or more different polypeptide.
4. Vernon Ingram showed the biochemical change to chain of sickle-cell hemoglobin is due to substitution of nonpolar amino acid valine for negatively charged amino acid glutamate.

D. From DNA to RNA to Protein

1. Classical geneticists conceived of a gene as any of the particles of inheritance on a chromosome.
2. To molecular geneticists, a gene is a sequence of DNA nucleotide bases that codes for a product.
3. DNA is restricted to nucleus; protein synthesis occurs in cytoplasm.
4. Ribonucleic acid (RNA) was found in both regions and was likely intermediary in protein synthesis.

E. Types of RNA

1. Like DNA, RNA is a polymer of necleotides.
2. Unlike DNA, RNA is single -stranded, contains the sugar ribose, and contains the base uracil instead of thymine.
3. There are three major classes of RNA.
1. Messenger RNA (mRNA) takes a message from DNA in nucleus to ribosomes in cytoplasm.
2. Ribosomal RNA (rRNA) and proteins make up ribosomes where proteins are synthesized.
3. Transfer RNA (tRNA) transfers a particular amino acid to a ribosome.

F. The Required Steps

1. DNA undergoes transcription to mRNA, which is translated to a protein.
2. DNA is a template for RNA formation during transcription.
3. Transcription is the first step in gene expression; it is the process whereby a DNA strand serves as a template for the formation of mRNA.
4. An mRNA transcript directs the sequence of amino acids in a polypeptide.

15.2. The Genetic Code

A. Sequence of Bases in DNA

1. The central dogma of molecular biology states that the sequence of nucleotides in DNA specifies the order of amino acids in a polypeptide.
2. The genetic code is a triplet code comprised of 64 three-base code words (codons).
3. Codon consists of 3 nucleotide bases of DNA.

B. Finding the Genetic Code

1. In 1961, Marshall Nirenberg and J. Heinrich Matthei found an enzyme that could be used to construct synthetic RNA; discovered the codon UUU coded for phenylalanine.
2. The code is degenerate; there are 64 triplets to code for 20 naturally occurring amino acids and this robustness protects against potentially harmful mutations.
3. The genetic code is unambiguous; each triplet codon has only one meaning.
4. The code has start and stop signals: there is one start codon and three stop codons.

C. The Code is Universal

1. The few exceptions to universality of the genetic code suggests code dates to very first organisms.
2. Once the code was established, changes would ve very disruptive.

15.3. The First Step: Transcription

A. Transcription

1. The first step required for gene expression; takes place in the nucleus of eukaryotic cells.
2. mRNA formation usually leads to a polypeptide gene product; however, tRNA and rRNA are also transcribed from DNA templates and are products themselves.
3. Enzymes called RNA polymerases are involved in transcription.

B. Messenger RNA is Formed

1. Transcription begins when RNA polymerase attaches to a promoter on DNA.
2. RNA polymerase is an enzyme that speeds formation of RNA from a DNA template.
3. Promoter is DNA region that defines start of gene, direction of transcription, and strand copied.
4. Next, a segment of the DNA helix unwinds and unzips.

C. RNA Polymerase

1. As RNA polymerase moves along the template strand of the DNA, complementary RNA nucleotides pair with DNA nucleotides of the strand.
2. RNA polymerase joins the RNA nucleotides together in the 5' --->3' direction.
3. Transcription begins when RNA polymerase attaches to a region of DNA called a promoter; a promoter defines the start of a gene, the direction of transcription, and the strand transcribed.
4. RNA/DNA association is not as stable as DNA helix; therefore, only newest portion of RNA molecule associated with RNA polymere is bound to DNA; the rest dangles off to side.
5. Elongation of mRNA continues until RNA polymerase comes to a DNA terminator sequence.
6. Terminator sequence causes RNA polymerase to stop transcribing DNA and to release mRNA transcript.
7. RNA polymerase molecules work to produce mRNA and from same DNA molecule at same time.
8. Cells produce thousands of copies of same mRNA molecule and many copies of coded protein in a shorter period of time if a single copy of DNA were used for protein synthesis.

D. Messenger RNA is Processed

1. In eukaryotes, newly formed primary mRNA transcript is processed before leaving nucleus.
2. Primary mRNA transcript is immediate product of transcription; contains exons and introns.
3. Ends of the mRNA molecule are altered: a cap is put on 5-end and a poly-A tail is put on 3-end.
1. "Cap" is a modified guanine (G) that tells a ribosome where to attach to begin translation.
2. The "poly-A tail" consists of a 150--200 adenine (A) nucleotide chain that facilitates transport of mRNA out of the nucleus and inhibits degradation of mRNA by enzymes.
4. Portions of the primary mRNA transcript, called introns, are removed.
1. Exon is portion of DNA code in primary mRNA transcript eventually expressed as a result of polypeptide synthesis.
2. Intron is non-coding segment of DNA removed by spliceosomes before mRNA leaves nucleus.
5. Spliceosomes are a complex that contains several kinds of ribonucleoproteins; it cuts primary mRNA
transcript and then rejoins adjacent exons.
6. Investigators have found that the simpler the eukaryote, the less likely that introns will be present.
7. Role of introns is being investigated: they may allow crossing over during meiosis, or introns may divide a gene into regions that can be joined in different combinations for different products; thyroid and pituitary glands process same primary mRNA transcript to produce different products.
8. An intron has been discovered in the gene for a tRNA molecule in the cyanobacterium Anabaena; this particular intron is "self-splicing" (it has capability of splicing itself out of an RNA transcript.)
9. Ribozymes are RNAs with an enzymatic function restricted to cleaving RNA at specific locations.
1. RNA could have served as both genetic material and as first enzymes in early life forms.
2. This hypothesis eliminates dilemma of which came first, DNA or protein; RNA came first.

15.4. The Second Step: Translation

A. Translation

1. Takes place in cytoplasm of eukaryotic cells.
2. Translation is a second step by which gene expression leads to protein synthesis.
3. One language (nucleic acids) is translated into another language (protein).

B. The Role of Transfer RNA

1. Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes.
2. tRNA is a single-stranded ribonucleic acid that doubles back on itself to create regions where complimentary bases are hydrogen-bonded to one another.
3. At the 3' end it binds to amino acid; at other end it has an anticodon that binds to mRNA codon; anticodon is group of nucleotides on tRNA complementary to codon on mRNA.
4. There is at least one tRNA molecule for each of the 20 amino acids found in proteins; there are fewer tRNA's that codons because some tRNA's pair with more than one codon.
5. tRNA synthetases are amino acid-activating enzymes that recognize which amino acid should join which tRNA molecule, and then catalyze ATP-requiring reactions joining them.
6. Amino acid-tRNA complex forms, then travels in cytoplasm to ribosome for protein synthesis.

C. The Role of Ribosomal

1. Ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of nucleus.
2. rRNA is packaged with a variety of proteins into ribosomal subunits, one larger than the other.
3. Subunits move separately through nuclear envelope pores into cytoplasm where they combine.
4. Ribosomes can float free in cytosol or attach to endoplasmic reticulum.
5. Prokaryotic cells contain about 10,000 ribosomes; eukaryotic cells contain many times more.
6. Ribosomes have a binding site for mRNA and binding sites for two transfer RNA (tRNA) molecules.
7. They facilitate complementary base pairing between tRNA anti-codons and mRNA codons; one protein is an enzyme that joins amino acids together by means of a peptide bond.
8. Ribosome move down mRNA molecule, new tRNA's arrive; amino acids join; polypeptide forms.
9. Translation terminates once polypeptide is formed; ribosome dissociates into its two subunits.
10. Polyribosomes are clusters of several ribosomes synthesizing the same protein.
11. Ribosomes are first free in cystol; once synthesis begins, some have series of amino acids called signal sequence that enables ribosome to bind to endoplasmic reticulum.
12. Nearly all polypeptide have signal sequences that target them for final location in cell.

D. Translation Requires three steps.

1. In translation, mRNA codons base-pair with tRNA anti-codons carrying specific amino acids.
2. Codon order determines order of tRNA molecules and sequence of amino acids in polypeptide.
3. Protein synthesis involves chain initiation, chain elongation, chain termination.
4. Enzymes are required for all three steps; energy is needed for first two steps.
5. Chain Initiation
1. Small ribosomal subunit attaches to mRNA in vicinity of the start codon: a base triplet (AUG).
2. First initiator tRNA pairs with this codon; then large ribosomal subunit joins to small subunit.
3. Each ribosome contains tow sites: the P (for peptide) site and the A (for amino acid) site.
4. Initiator tRNA binds to P site although it carries one amino acid; the A site is for next tRNA.
5. Initiation factor proteins are required to bring necessary translation components together.
6. Chain elongation
1. tRNA with attached polypeptide is at P site; tRNA -- amino acid complex is just arriving at A site.
2. The polypeptide is transferred and attached by a peptide bond to the newly arrived amino acid.
3. Reaction is catalyzed by a ribozyme, which is part of the larger subunit.
4. The tRNA molecule in the P site leaves.
5. Translocation occurs with mRNA, along with peptide-bearing tRNA, moves from site A to P.
6. As ribosome has moved forward three nucleotides, there is new codon located at empty A site.
7. The complete cycle is rapidly repeated, about 15 times per second in Escherichia coli.
7. Chain Termination
1. Termination of polypeptide synthesis occurs at stop codon that does not code for amino acid.
2. The polypeptide is enzymatically cleaved from the last tRNA.
3. tRNA and polypeptide leave the ribosome, which dissociates into its two subunits.