From DNA to Protein: (Central Dogma). From DNA to Protein: Genotype to Phenotype. One Gene, One Polypeptide DNA, RNA, and the Flow of Information Transcription: DNA-Directed RNA Synthesis The Genetic Code Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes
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From DNA to Protein: Genotype to Phenotype • One Gene, One Polypeptide • DNA, RNA, and the Flow of Information • Transcription: DNA-Directed RNA Synthesis • The Genetic Code • Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • Translation: RNA-Directed Polypeptide Synthesis • Regulation of Translation • Posttranslational Events • Mutations: Heritable Changes in Genes
One Gene, One Polypeptide • A gene is defined as a DNA sequence. • There are many steps between genotype and phenotype; genes cannot by themselves produce a phenotype.
One Gene, One Polypeptide • In the 1940s, Beadle and Tatum showed that when an altered gene resulted in an altered phenotype, that altered phenotype always showed up as an altered enzyme. • They experimented with strains of the bread mold Neurospora: a wild-type, and several mutant strains. • Their results suggested that mutations cause a defect in only one enzyme in a metabolic pathway. • This lead to the one-gene, one-enzyme hypothesis.
One Gene, One Polypeptide • The gene–enzyme connection has undergone several modifications. Some enzymes are composed of different subunits coded for by separate genes. • This suggests, instead of the one-gene, one enzyme hypothesis, a one-gene, one-polypeptide relationship.
DNA, RNA, and the Flow of Information • The expression of a gene takes place in two steps: • Transcription makes a single-stranded RNA copy of a segment of the DNA. • Translation uses information encoded in the RNA to make a polypeptide.
DNA, RNA, and the Flow of Information • RNA (ribonucleic acid) differs from DNA in three ways: • RNA consists of only one polynucleotide strand. • The sugar in RNA is ribose, not deoxyribose. • RNA has uracil instead of thymine. • RNA can base-pair with single-stranded DNA (adenine pairs with uracil instead of thymine) and also can fold over and base-pair with itself.
DNA, RNA, and the Flow of Information • Francis Crick’s central dogma stated that DNA codes for RNA, and RNA codes for protein. • How does information get from the nucleus to the cytoplasm? • What is the relationship between a specific nucleotide sequence in DNA and a specific amino acid sequence in protein?
DNA, RNA, and the Flow of Information • Messenger RNA, or mRNA moves from the nucleus of eukaryotic cells into the cytoplasm, where it serves as a template for protein synthesis. • Transfer RNA, or tRNA, is the link between the code of the mRNA and the amino acids of the polypeptide, specifying the correct amino acid sequence in a protein.
DNA, RNA, and the Flow of Information • Certain viruses use RNA rather than DNA as their information molecule during transmission. • These viruses transcribe from RNA to RNA; they make a complementary RNA strand and then use this “opposite” strand to make multiple copies of the viral genome by transcription. • HIV and certain tumor viruses (called retroviruses) have RNA as their infectious information molecule; they convert it to a DNA copy inside the host cell and then use it to make more RNA.
Transcription: DNA-Directed RNA Synthesis • In normal prokaryotic and eukaryotic cells, transcription requires the following: • A DNA template for complementary base pairing • The appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as substrates • The enzyme RNA polymerase
Transcription: DNA-Directed RNA Synthesis • Just one DNA strand (the template strand) is used to make the RNA. • For different genes in the same DNA molecule, the roles of these strands may be reversed. • The DNA double helix partly unwinds to serve as template. • As the RNA transcript forms, it peels away, allowing the already transcribed DNA to be rewound into the double helix.
Transcription: DNA-Directed RNA Synthesis • The first step of transcription, initiation, begins at a promoter, a special sequence of DNA. • There is at least one promoter for each gene to be transcribed. • The RNA polymerase binds to the promoter region when conditions allow. • The promoter sequence directs the RNA polymerase as to which of the double strands is the template and in what direction the RNA polymerase should move.
Transcription: DNA-Directed RNA Synthesis • After binding, RNA polymerase unwinds the DNA about 20 base pairs at a time and reads the template in the 3¢-to-5¢ direction (elongation). • The new RNA elongates from its 5¢ end to its 3¢ end; thus the RNA transcript is antiparallel to the DNA template strand. • Transcription errors for RNA polymerases are high relative to DNA polymerases.
Transcription: DNA-Directed RNA Synthesis • Particular base sequences in the DNA specify termination. • Gene mechanisms for termination vary: • For some, the newly formed transcript simply falls away from the DNA template. • For other genes, a helper protein pulls the transcript away. • In prokaryotes, translation of the mRNA often begins before transcription is complete.
The Genetic Code • A genetic code relates genes (DNA) to mRNA and mRNA to the amino acids of proteins. • mRNA is read in three-base segments called codons. • The number of different codons possible is 64 (43), because each position in the codon can be occupied by one of four different bases. • The 64 possible codons code for only 20 amino acids and the start and stop signals.
The Genetic Code • AUG, which codes for methionine, is called the start codon, the initiation signal for translation. • Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes to end translation.
The Genetic Code • After subtracting start and stop codons, the remaining 60 codons code for 19 different amino acids. • This means that many amino acids have more than one codon. Thus the code is redundant. • However, the code is not ambiguous. Each codon is assigned only one amino acid.
The Genetic Code • In the early 1960s, molecular biologists broke the genetic code. • Nirenberg prepared an artificial mRNA in which all bases were uracil (poly U). • When incubated with additional components, the poly U mRNA led to synthesis of a polypeptide chain consisting only of phenylalanine amino acids. • UUU appeared to be the codon for phenylalanine. • Other codons were deciphered from this starting point.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The molecule tRNA is required to assure specificity in the translation of mRNA into proteins. • The tRNAs must read mRNA correctly. • The tRNAs must carry the correct amino acids.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The codon in mRNA and the amino acid in a protein are related by way of an adapter—a specific tRNA molecule. • tRNA has three functions: • It carries an amino acid. • It associates with mRNA molecules. • It interacts with ribosomes.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • A tRNA molecule has 75 to 80 nucleotides and a three-dimensional shape (conformation). • The shape is maintained by complementary base pairing and hydrogen bonding. • The three-dimensional shape of the tRNAs allows them to combine with the binding sites of the ribosome.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • At the 3¢ end of every tRNA molecule is a site to which its specific amino acid binds covalently. • Midpoint in the sequence are three bases called the anticodon. • The anticodon is the contact point between the tRNA and the mRNA. • The anticodon is complementary (and antiparallel) to the mRNA codon. • The codon and anticodon unite by complementary base pairing.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • Each ribosome has two subunits: a large one and a small one. • In eukaryotes the large one has three different associated rRNA molecules and 45 different proteins. • The small subunit has one rRNA and 33 different protein molecules. • When they are not translating, the two subunits are separate.
Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The small ribosomal subunit plays a role in validating the three-base-pair match between the mRNA and the tRNA. • If hydrogen bonds have not formed between all three base pairs, the tRNA is ejected from the ribosome.
Translation: RNA-Directed Polypeptide Synthesis • Translation begins with an initiation complex: a charged tRNA with its amino acid and a small subunit, both bound to the mRNA. • This complex is bound to a region upstream of where the actual reading of the mRNA begins. • The start codon (AUG) designates the first amino acid in all proteins. • The large subunit then joins the complex. • The process is directed by proteins called initiation factors.
Translation: RNA-Directed Polypeptide Synthesis • Ribosomes move in the 5¢-to-3¢direction on the mRNA. • The peptide forms in the N–to–C direction. • The large subunit catalyzes two reactions: • Breaking the bond between the tRNA in the P site and its amino acid • Peptide bond formation between this amino acid and the one attached to the tRNA in the A site • This is called peptidyl transferase activity.
Translation: RNA-Directed Polypeptide Synthesis • After the first tRNA releases methionine, it dissociates from the ribosome and returns to the cytosol. • The second tRNA, now bearing a dipeptide, moves to the P site. • The next charged tRNA enters the open A site. • The peptide chain is then transferred to the P site. • These steps are assisted by proteins called elongation factors.
Translation: RNA-Directed Polypeptide Synthesis • When a stop codon—UAA, UAG, or UGA—enters the A site, a release factor and a water molecule enter the A site, instead of an amino acid. • The newly completed protein then separates from the ribosome.
Regulation of Translation • Antibiotics are defensive molecules produced by some fungi and bacteria, which often destroy other microbes. • Some antibiotics work by blocking the synthesis of the bacterial cell walls, others by inhibiting protein synthesis at various points. • Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are unaffected.
Regulation of Translation • Polysomes are mRNA molecules with more than one ribosome attached. • These make protein more rapidly, producing multiple copies of protein simultaneously.
Posttranslational Events • Two posttranslational events can occur after the polypeptide has been synthesized: • The polypeptide may be moved to another location in the cell, or secreted. • The polypeptide may be modified by the addition of chemical groups, folding, or trimming.
Figure 12.14 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
Posttranslational Events • As the polypeptide chain forms, it folds into its 3-D shape. • The amino acid sequence also contains an “address label” indicating where in the cell the polypeptide belongs. It gives one of two sets of instructions: • Finish translation and be released to the cytoplasm. • Stall translation, go to the ER, and finish synthesis at the ER surface.