12: From DNA to Protein: Genotype to Phenotype

One Gene, One Polypeptide

· There are many steps between genotype and phenotype; genes cannot by themselves directly produce a phenotype.

· A gene is defined as a DNA sequence.

· In the 1940s, Beadle and Tatum showed that an altered gene resulted in an altered phenotype that showed up as an altered enzyme.

· They used the bread mold Neurospora crassa.

· This is an organism with a haploid vegetative life cycle, so recessive mutations are easy to detect.

· Neurospora were grown on minimal medium consisting of just sucrose, minerals, and a few vitamins.

· Wild-type Neurospora were treated with a mutagen, an agent that causes changes in the DNA.

· After treatment, they were grown in a complete medium.

· When testing some of the treated strains, some were found that could no longer grow on minimal medium, but instead needed certain supplements.

· These nutrient-requiring auxotrophs were assumed to have mutated.

· For each auxotrophic strain, Beadle and Tatum were able to find a single compound that could support its growth.

· One group of mutants needed arginine to grow.

· Mapping studies established that some of these arg mutations are at different loci, and therefore are in different genes.

· Beadle and Tatum demonstrated that these different mutants had defective genes for the same biochemical pathway, the pathway leading to arginine synthesis. (See Figure 12.1.)

· If the gene defect affected earlier enzyme steps in the pathway, several different substances could substitute for arginine.

· If the defect was for the enzyme step just before arginine synthesis, only arginine could substitute.

· Beadle and Tatum thus postulated the one gene, one enzyme hypothesis.

· Later it was learned that some enzymes are composed of different subunits coded for by separate genes.

· The one gene, one enzyme hypothesis was later changed to the one-gene, one-polypeptide hypothesis.

· Even this hypothesis requires modification because some genes code for RNA molecules that are never translated into polypeptides.

DNA, RNA, and the Flow of Information

· Two steps are used to express a gene:

· Transcription makes a single-stranded RNA copy of a segment of the DNA.

· Translation uses information encoded in a portion of the RNA to make a polypeptide.

· In eukaryotes, these two steps are physically separated.

RNA differs from DNA

· RNA is single-stranded.

· The sugar in RNA is ribose, not deoxyribose.

· Wherever thymine is found in DNA, it is replaced by uracil in RNA.

· RNA can fold over and base-pair with itself.

Information flows in one direction when genes are expressed

· Francis Crick's central dogma stated that DNA codes for RNA, and RNA codes for protein; that is, once information passes into protein, it cannot get out again. (See Figure 12.2a.)

· Messenger RNA (mRNA) moves from the nucleus of eukaryotic cells into the cytoplasm where it serves as a template for protein synthesis.

· Transfer RNA (tRNA) is the link between the code of the mRNA and the amino acids of the polypeptide.

· The tRNA molecules specify the correct amino acid. (See Figure 12.3.)

RNA viruses modify the central dogma

· RNA viruses are viruses that use RNA as their information molecule during transmission.

· Examples are the influenza virus and poliovirus.

· HIV and certain tumor viruses have RNA as their infectious information molecule, but convert it to a DNA copy inside the host cell, then use it to make more RNA.

· See Figure 12.2b for an illustration of retroviruses that are capable of this reverse transcription.

Transcription: DNA-Directed RNA Synthesis

· RNA polymerase is the enzyme that uses DNA as a template to make RNA.

· Just one of the strands of a gene’s DNA is used to make the RNA.

· This strand is called the template strand and is used for transcription.

· The other untranscribed strand is called the complementary strand.

· For different genes in the same DNA molecule, however, the roles of these strands may be reversed.

· The continuous double helix of DNA has many regions that are read by RNA polymerase

· 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.

Initiation of transcription requires a promoter and an RNA polymerase

· Transcription of a gene begins at a promoter, which is a certain sequence of DNA.

· There is at least one promoter for each gene to be transcribed into mRNA.

· 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.

· In effect, the promoters serve as punctuation marks for the transcription process.

· RNA is synthesized in the 5¢-to-3¢ direction, moving along the template DNA in the 3¢-to-5¢ direction. (See Figure 12.4.)

· Not all promoters are identical. Some bind RNA polymerase more effectively; this causes them to be transcribed more frequently, when other conditions allow.

· Prokaryotes have one type of RNA polymerase that transcribes mRNA, tRNA, and rRNA.

· Eukaryotes have three different RNA polymerases: RNA polymerase I, II, and III.

· RNA polymerase II makes all mRNA in eukaryotes.

· In eukaryotic cells, other proteins must bind to the DNA around the promoter to prepare a “docking site.”

· This eukaryotic feature allows a way to regulate the transcription of particular genes.

RNA polymerase elongates the transcript

· After binding, RNA polymerase unwinds the DNA about 20 base pairs at a time and reads the template in the 3¢-to-5¢ direction.

· The RNA transcript is antiparallel to the DNA template strand.

· Energy for synthesis comes from the removal and breakdown of the pyrophosphate group from each nucleoside added.

· Transcription errors for RNA polymerases are high relative to DNA polymerases; a mistake occurs for every 10⁴ to 10⁵ bases incorporated.

· These are errors in the copies, however, not the original DNA master, so they are less likely to be harmful.

Transcription terminates at particular base sequences

· Particular base sequences in the DNA specify termination.

· Gene mechanisms for termination vary.

· For some genes, the newly formed transcript simply falls away from the DNA template and the RNA polymerase.

· For other genes, a helper protein pulls the transcript away.

· In prokaryotes, translation of the mRNA often begins before transcription is complete.

· In eukaryotes, the process is more complicated and involves a spatial separation as well as further processing.

The Genetic Code

· DNA codes for RNA by the transcription process.

· mRNA is read in three-base contiguous segments called codons.

· The number of different codons possible is 64, because each position in the codon can be occupied by one of four different bases.

· Four possibilities for the first base, times four for the second, times four for the third yields 64 possibilities.

· The 64 possible codons code for only 20 amino acids and the start and stop signals found in all mRNA molecules.

· AUG, which codes for methionine, is called the start codon. The start codon initiates translation.

· Three of the possible codons are stop codons (UAA, UAG, and UGA).

· Stop codons direct the ribosomes to stop reading the mRNA; that is, they end translation.

The genetic code is redundant but not ambiguous

· 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.

· But the code is not ambiguous. Each codon is assigned only one amino acid, not two or three possible amino acids.

· The tRNA molecules that have the correct amino acids attached determine the assignment of amino acids to the mRNA codons.

· The genetic code is nearly universal, applying to all species on our planet. (See Figure 12.5.)

· Minor variations are found within mitochondria and chloroplasts; other exceptions are few and slight.

· This common genetic code has great implications in genetic engineering.

Biologists broke the genetic code by using artificial messengers

· Decoding breakthroughs started in 1961.

· Early on, the likelihood of a three-letter codon was postulated.

· Nirenberg prepared an artificial mRNA in which all bases were uracil.

· Incubated with required 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.

· An additional technique finished the deciphering.

· Simple synthetic mRNA’s, three nucleotides long, could bind to ribosomes.

· This complex then caused the tRNA-amino acid to bind according to the three-letter codon.

· Using this technique, the code was fully deciphered. (See Figure 12.6.)

· Radioactive labeling was also used to decipher the code.

Preparation for Translation: Linking RNA’s, Amino Acids, and Ribosomes

· Translation occurs at ribosomes, which are molecular protein synthesizing machines that hold mRNA and tRNA in place.

· In prokaryotes, ribosomes bind to mRNA as it is being synthesized.

· In eukaryotes, mRNA is made in the nucleus, and translation occurs at the ribosomes in the cytoplasm.

· To assure protein specificity:

· The tRNA’s must read mRNA correctly.

· The tRNA’s must carry the correct amino acids.

Transfer RNA’s carry specific amino acids and bind to specific codons

· Specific tRNA molecules function as adapters.

· Each carries an amino acid, associates with mRNA molecules, and interacts with ribosomes.

· A tRNA molecule has 75 to 80 nucleotides.

· It has a three-dimensional shape maintained by complementary base pairing and hydrogen bonding. (See Figure 12.7. The Instructor’s Resource CD-ROM includes a space-filling model of tRNA.)

· 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.

· There are fewer anticodon codes than mRNA codons.

· This is possible because some codon-anticodon interactions tolerate a mismatch at the 3¢ base of the mRNA. (See Figure 12.5.)

· This is called wobble, but does not allow the genetic code to be ambiguous.

· The three-dimensional shape of the tRNA’s allows them to combine with the binding sites of the ribosome.

Activating enzymes link the right tRNA’s and amino acids

· The correct amino acids are attached to the correct tRNA’s by a family of activating enzymes called aminoacyl-tRNA synthetases.

· Each activating enzyme is specific for one amino acid and its tRNA.

· The enzyme has a three-part active site that binds a specific amino acid, ATP, and a specific tRNA, which is charged with a high-energy bond.

· This bond provides the energy for making the peptide band that will join adjacent amino acids.

· The reactions have two steps (see Figure 12.8):

· Enzyme + ATP + AA ® enzyme—AMP—AA + PP_i

· Enzyme—AMP—AA + tRNA ® enzyme + AMP + tRNA—AA

The ribosome is the staging area for translation

· Each ribosome has two subunits, a larger one and a smaller one. (See Figure 12.9.)

· The large one in eukaryotes has three different associated rRNA molecules and 45 different proteins.

· The smaller subunit has one rRNA and 33 different protein molecules.

· When they are not translating, the two subunits are separate.

· Ribosomes of prokaryotes are somewhat smaller, and their ribosomal proteins and rRNA’s are different.

· The different proteins and rRNA’s are held together by ionic bonds and hydrophobic forces, not covalent bonds.

· The structure can self-assemble if disassembled by detergents.

· Ribosomes are simply molecular factories and are nonspecific. They combine with any mRNA and all tRNA’s.

· The large subunit has four sites where tRNA molecules bind. (See Figure 12.9.)

· The T site is where the tRNA first lands. It is brought to the site by a special protein, the T, or transfer, factor.

· The A site is where the tRNA anticodon binds to the mRNA codon.

· The P site is where the tRNA adds its amino acid to the growing polypeptide chain.

· The E (exit) site is where the tRNA, less its amino acid, goes before leaving the ribosome.

Translation: RNA-Directed Polypeptide Synthesis

Translation begins with an initiation complex

· At the beginning of translation, an initiation complex forms.

· The initiation complex includes the first tRNA and its amino acid, a small subunit of the ribosome, and an mRNA molecule. (See Figure 12.10.)

· This complex is bound to a region upstream (toward the 5¢ end) of where the actual reading of the mRNA begins.

· The start codon (AUG) for methionine designates the first amino acid in all proteins. (However, some proteins are trimmed after synthesis, and the methionine is thereby removed.)

· The large subunit then joins the complex.

· The process is directed by proteins called initiation factors, which use GTP as an energy source.

The polypeptide elongates from the N terminus

· Ribosomes move in the 5¢-to-3¢ direction on the mRNA. (See Figure 12.11.)

· They synthesize the peptide in the N terminus-to-C terminus direction.

· The large subunit catalyzes two reactions:

· Breakage of the bond between the tRNA in the P site and its amino acid (on the polypeptide).

· Peptide bond formation between this (tRNA-attached) amino acid and the tRNA in the A site.

· This is called peptidyl transferase activity.

· One of the rRNA’s in the large subunits appears to participate in the catalysis of this reaction.

· Here we see rRNA acting as the catalyst, or ribozyme.

Elongation continues and the polypeptide grows

· The first tRNA releases methionine, dissociates from the ribosome, and returns to the cytosol.

· The second tRNA then 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.

A release factor terminates translation

· 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. (See Figure 12.12.)

· The newly completed protein then separates from the ribosome.

Regulation of Translation

Some antibiotics work by inhibiting translation

· Antibiotics are defense molecules.

· They are produced by some fungi and bacteria.

· They have been used to combat human bacterial infectious disease.

· Antibiotics must specifically destroy microbial invaders, but not harm the human host.

· Some antibiotics work by blocking the synthesis of the bacterial cell walls, others by inhibiting protein synthesis. (See Table 12.2.)

· Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are unaffected.

Polysome formation increases the rate of protein synthesis

· Polysomes are mRNA molecules with more than one ribosome attached. (See Figure 12.13.)

· These make protein more rapidly, producing multiple copies of protein simultaneously.

Posttranslational Events

· Some proteins require additional modification after synthesis before they become functional.

· New chemical groups might be added to the protein, it might be folded (with the assistance of other proteins), or it might get trimmed.

Chemical signals in proteins direct them to their cellular destinations

· See Figure 12.14 for an illustration of the possible destinations for a newly translated protein.

· As the polypeptide chain forms, it spontaneously folds into its three-dimensional shape.

· The amino acid sequence also contains an “address label” indicating where in the cell the polypeptide belongs.

· All protein synthesis begins on free ribosomes in the cytoplasm.

· In eukaryotes, as the peptide chain is made, information on the nascent portion gives one of two sets of instructions:

· Finish translation and be released to the cytoplasm.

· Stall translation, go to the endoplasmic reticulum, and finish synthesis at the ER surface.

· Those destined to finish synthesis in the cytoplasm may contain information in their amino acid sequence that specifies where they belong: the nucleus, mitochondria, or peroxisomes.

· Some of the proteins that are transported to a destination require chaperonin proteins and docking proteins at the membrane that the protein must cross to its organelle destination.

· Those destined for the ER generate an approximately 25-amino-acid-long hydrophobic leader sequence that signals to a signal recognition particle, which is composed of protein and RNA. (See Figure 12.15.)

· The association of the signal to the signal receptor particle stalls any additional translation.

· This stall continues until the ribosome attaches to a specific receptor protein on the surface of the ER.

· Translation continues with the protein moving through a pore in the ER membrane.

· Some proteins have signals that direct the embedding of the protein into the ER membrane.

· This is when membrane proteins of the ER, Golgi apparatus, lysosomes, and plasma membrane get positioned.

· Other signals direct the protein to the Golgi apparatus, lysosomes, or to the outside of the cell.

· Proteins with no signals from the ER go through the Golgi apparatus and are secreted from the cell.

Many proteins are modified after translation

· It is the exception, not the rule, that the finished protein is identical to the translation from the mRNA code.

· Modifications are often essential to the final functioning of the protein. (See Figure 12.16.)

· Proteolysis is the cleavage of the protein to make a shortened finished protein.

· Insulin is an example of a protein that gets trimmed.

· The signal to go to the ER is often cleaved after the protein gets there.

· The virus HIV needs a protease to cleave a protein. One treatment for HIV inhibits this enzyme.

· Glycosylation involves the addition of sugars to the protein.

· Signals in the amino acid sequence of the protein direct the addition of the sugars in the ER by resident enzymes.

· Additional modifications occur in both the Golgi apparatus and the ER.

· Phosphorylation is the addition of phosphate groups to certain proteins. These additions may be temporary and affect the activity of the protein by changing the three-dimensional shape.

Mutations: Heritable Changes in Genes

· Mutations are heritable changes in DNA—changes that are passed on to daughter cells.

· In single-celled organisms, any mutations that occur are passed to the daughter cells at the time of cell division.

· Multicellular organisms have two types of mutations:

· Somatic mutations are passed on during mitosis, but the affected cells never become gametes and so do not pass to subsequent generations.

· Germ-line mutations are mutations that occur in cells that might give rise to gametes.

· Some mutations cause visible phenotypic change. Others cause metabolic changes that might not yet be detectable.

· Some mutations exert their effect only under certain restrictive conditions.

· These are called conditional mutants.

· They are unaffected under permissive conditions, but express the mutant phenotype at the restrictive condition.

· Temperature-sensitive mutants are an example.

· All mutations are alterations of the DNA nucleotide sequence and are of two types:

· Point mutations are mutations of single genes.

· Chromosomal mutations are changes in the arrangements of chromosomal DNA segments.

Point mutations are changes in single bases

· Point mutations result from the addition or subtraction of a nucleotide base or the substitution of one base for another.

· Point mutations can occur as a result of mistakes during DNA replication, or by environmental mutagens, such as chemicals and radiation.

· Because of redundancy in the genetic code, some point mutations result in no change in the amino acids in the protein.

· These are called silent mutations.

· Some mutations cause an amino acid substitution.

· These are called missense mutations.

· An example in humans is sickle-cell anemia, a defect in the b-globin subunits of hemoglobin. (The Instructor’s Resource CD-ROM includes light micrographs of normal and sickled red blood cells.)

· The red blood cells collapse when oxygen levels are low.

· Missense mutations might reduce the functioning of a protein or disable it completely.

· Nonsense mutations are base substitutions that cause a change from a codon that instructs the incorporation of an amino acid to a codon that terminates translation.

· A frame-shift mutation is when a single base is inserted or deleted in a gene.

· This causes the most disruption when the event occurs at or near the beginning of the template.

· This type of mutation shifts the code, changing many of the codons to different codons.

· These shifts almost always lead to the production of nonfunctional proteins.

Chromosomal mutations are extensive changes in the genetic material

· DNA molecules can break and re-form.

· This can cause four different types of mutations: deletions, duplications, inversions, and translocations. (See Figure 12.18.)

· Deletions are a loss of a chromosomal segment.

· Duplications are a repeat of a segment.

· Breaking and rejoining leads to inversions if segments get reattached in the opposite orientation.

· Translocations result when a portion of one chromosome attaches to another.

· Translocations can be reciprocal (see Figure 12.18d) or nonreciprocal.

· Translocations can make synapses in meiosis difficult and can lead to aneuploidy (too many or too few chromosomes).

Mutations can be spontaneous or induced

· Spontaneous mutations are permanent changes that occur without outside influence. (see Figure 12.19)

· Spontaneous mutations may be caused by any of several mechanisms.

· Nucleotides occasionally change their structure (called a tautomeric shift).

· A base may temporarily change to its unusual tautomer at the same time that replication is occurring.

· The tautomer may pair with the alternate purine if it is a purine, or the alternate pyrimidine if it is a pyrimidine.

· DNA polymerase sometimes makes errors in replication.

· These errors are often repaired by the proofreading function of the replication complex, but some errors escape and become permanent.

· Meiosis is imperfect. Nondisjunction can occur. Random chromosome breaks rejoin incorrectly, leading to translocations.

· Induced mutations are permanent changes caused by some outside agent.

· Some chemicals alter covalent bonds in nucleotides.

· Nitrous acid deaminates cytosine, converting it to uracil.

· DNA polymerase mistakes uracil for thymine and puts an A in during replication instead of the G that would have been incorporated otherwise.

· Benzoapyrene, a product of incomplete combustion, which is found in all smoke, adds a large chemical group to guanine, making it unavailable for base pairing.

· Any base might be inserted to fill the gap.

· Radiation damages DNA.

· Ionizing radiation (X rays) produces highly reactive compounds and atoms called free radicals.

· Gamma rays also produce free radicals.

· Free radicals can alter bases or break the sugar–phosphate backbone, causing chromosomal abnormalities.

· Ultraviolet radiation is absorbed by pyrimidines in the DNA, and when two thymines or two cytosines are next to each other on the same strand of a double-stranded DNA molecule, a covalent bond can form.

· Their interstrand covalent bonds make the DNA unreplicable.

· The long-term benefit of mutations is that they provide a genetic diversity for evolution and account for all the differences between and within organisms, excluding the effect of different environments.

· The detriment of mutation is the outright death or poor fit of an organism to its environment.

Mutations are the raw material of evolution

· Mutations are rare events and most of them are point mutations involving one nucleotide.

· Frequency of mutations is much lower than one mutation per 10⁴ genes per DNA duplication. Sometimes they are as rare as one per 10⁹ genes per duplication.

· Different organisms vary in mutation frequency.

· Mutations can be detrimental, neutral, or occasionally beneficial.

· Humans have 1,000 times the DNA of a prokaryote.

· This is at least partially due to duplication of DNA sequences, and then to divergence of the sequences over time.

· Random accumulation of mutations in the extra copies of genes can lead to the production of new useful proteins.

The Instructor’s Resource CD-ROM includes a space-filling model of a tRNA, and light micrographs of normal and sickled red blood cells.