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From Gene to Protein (Chapter Seventeen)
THE CONNECTION BETWEEN GENES AND PROTEINS The Study of Metabolic Defects Provided Evidence That Genes Specify Proteins In 1909, Archibald Garrod suggested that genes dictated phenotypes through enzymes that catalyze specific chemical reactions in the cell. e
How Genes Control Metabolism: One Gene-One Enzyme Gene
Research conducted several years later supported Garrod’s idea that a gene dictates the production of a specific enzyme. Biochemists found that cells synthesize and degrade most organic molecules via metabolic pathways, in which each chemical reaction is catalyzed by a specific enzyme. A breakthrough in demonstrating the relationship between genes and enzymes occurred when Beadle and Edward Tatum began working with a bread mold. After treating the mold with X with X-rays, they looked for mutants that differed from the wild-type in their nutritional needs. Wild-type bread mold can wild type survive in the laboratory on agar mixed with inorganic salts, glucose, and biotin. They identified mutants that needed certain essential molecules because they couldn’t synthesize it themselves. They found that their mutants fell into three classes, each mutated in a different gene. In the pathway leading to synthesis of arginine, it was necessary to convert a precursor nutrient to orthinine, which is necessary converted to citrulline, which is converted to arginine. When they tested the mutants, their results showed that the mutants were blocked at different steps in the pathway. Since each mutant had one defective gene, their results provided strong support for the one gene-one enzyme hypothesis. one
One Gene-One Polypeptide
Researchers later learned that not all proteins are enzymes. Since proteins that are not enzymes are still gene products, molecular biologists began to think in terms of one gene one protein. However, ecular gene-one many proteins are made of two or more different polypeptide chains, with each polypeptide made by its own gene. Beadle and Tatum’s idea can then be restated as the one gene-one polypeptide genepolypeptide hypothesis. hypothesis Transcription and Translation Are the Two Main Processes Linking Gene to Protein: An Overview Recall that RNA contains ribose instead of deoxyribose, has the base uracil instead of thymine, and is usually single-stranded. Transcript ion is the synthesis of RNA under the direction of DNA. Just as a Transcription DNA strand provides a template for DNA synthesis of a complementary strand, it can provide a template for assembling a sequence of RNA nucleotides. This type of RNA molecule is called messenger mRNA), messenger RNA (mRNA), because it carries a message from the DNA. mRNA
2 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece) Translation is the actual synthesis of a polypeptide, which occurs under the direction of mRNA. During this stage, the cell translates the base sequence of mRNA into the amino acid sequence of a polypeptide. Ribosomal RNA serves as the translation site. In prokaryotes, DNA transcription and translation both take place in the cytosol. Thus, polypeptides can be synthesized during translation. In a eukaryotic cell, the nuclear envelope separates transcription from translation, which allows for RNA processing to yield the finished mRNA. An initial RNA transcript is the primary transcript transcript. In the Genetic Code, Nucleotide Triplets Specify Amino Acids Biologists established that triplets of nucleotide bases are the smallest units of uniform length that can code for all 20 amino acids. If each arrangement of three consecutive bases specifies an amino acid, there can be 64 possible code words. Experiments have verified that there is a triplet code genetic instructions for a code: polypeptide gene are written in the DNA as a series of three-nucleotide words. The intermediate step in translating a gene into amino acids is transcription, where the gene determines the sequence of base triplets along the length of an mRNA molecule. For each gene, the template strand of DNA is described (this strand is also known as the antisense strand, because the RNA produced will be complementary and exactly like the sense strand). The mRNA base triplets are called codons Each codon stands for one of the 20 amino acids or it could codons ns. be a stop codon that codes for a release factor. During translation, the sequence of codons are read in the 5’ → 3’ direction along the mRNA.
Cracking the Genetic Code
The first codon was deciphered in 1961 by Marshall Nirenberg, of the National Institutes of Health. He synthesized an artificial mRNA of all uracils to a test-tube mixture containing amino acids and ribosomes. His artificial system then created a polypeptide containing a single amnio acid, phenalanine (Phe). All 64 codons were then deciphered by the mid 1960s. The codon AUG is the start codon, while also coding for methionine (Met). UAA, UAG, and UGA are “stop” signals that mark the end of translation. While several codons can code for one amino acid, one codon never codes for more than one amino acid. The correct reading frame is necessary to be able to create the right nucleotides as read from 5’ → 3’ in groups of three.
3 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece) The Genetic Code Must Have Evolved Very Early In the History of Life The genetic code is nearly universal. Genes can be transcribed and translated after being transplanted from one species to another. Bacteria can be programmed by the insertion of human genes to synthesize certain human proteins that have important medical uses. Exceptions to this universality occur in certain single=celled eukaryotes, as well as certain mitochondria and chloroplasts. However, the near universality indicates that the genetic code must have existed very early in the history of life. THE SYNTHESIS AND PROCESSING OF RNA Transcription is the DNA-Directed Synthesis of RNA: A Closer Look mRNA, the carrier of information from DNA to the cell’s protein-synthesizing machinery, is transcribed from the emplate strand of a gene. RNA polymerase keeps the two strands of DNA apart and hooks together the RNA nucleotides as the base-pair along the DNA template. Like the DNA polymerases that function in DNA replication RNA polymerases can add nucleotides only to the 3’ end of the original strand. Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends. The DNA sequence where RNA polymerase attaches and initiates transcription is known as the promoter; the sequence that signals the end of transcription is the terminator The stretch of DNA terminator. to be transcribed into an RNA molecules is called a transcription unit unit.
RNA Polymerase Binding and Initiation of Transcription
The promoter of a gene includes within it the transcription start point and extends several dozen nucleotide pairs “upstream.” The promoter also serves as the binding site for RNA polymerase and determines which of the two strands of the DNA helix is used as the template. In prokaryotes, the RNA polymerase itself specifically recognizes and binds to the promoter. In eukaryotes, a collection of proteins called transcription factors mediate binding of RNA polymerase and the initiation of transcription. RNA polymerase only binds to the promoter after certain transcription factors are attached to it. The completed assembly of transcription factors and RNA polymerase bound to the promoter is called a transcription initiation complex The TATA box is a complex. nucleotide sequence containing TATA (on the sense strand) that is about 25 nucleotides upstream from the start point. A transcription factor that recognizes the TATA box will then bind to the DNA before RNA polymerase does so. Additional transcription factors will join the polymerase on the DNA to form the transcription initiation complex.
4 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece)
Elongation of the RNA Strand
As RNA polymerase moves along the DNA, it continues untwisting the double helix to allow pairing with RNA nucleotides. The DNA double helix re-forms and the new RNA molecule separates from the DNA template. A single gene can be transcribed simultaneously by several molecules of RNA polymerase following each other. Cells can then make the encoded protein in large amounts.
Termination of Transcription
Once the RNA polymerase reaches a terminator sequence in the DNA, transcription will end. In the prokaryotic cell, transcription stops right at the end of the termination signal, releasing both the DNA and RNA. In the eukaryotic cell, the polymerase continues for hundreds of nucleotides past the termination signal and then cuts off. Eukaryotic Cells Modify RNA after Transcription
Alteration of mRNA Ends
The 5’ end of a pre-mRNA molecule is capped off with a modified form of a guanine nucleotide (guanosine triphosphate). This 5’ cap helps protect the mRNA from degration by hydrolytic enzymes in the cytosol, and also functions as an attachment marker for ribosomes. The 3’ end of mRNA receives a poly(A) tail consisting of some 50 to 250 adenine nucleotides. This tail also inhibits degradation of the RNA and probably helps ribosomes to attach to it.
Split Genes and RNA Splicing
Large portions of the primary transcript are removed in RNA splicing Most eukaryotic genes and their splicing. RNA transcripts have long noncoding stretches of nucleotides that are interspersed between coding segments of the gene. The noncoding segments are called intervening sequences or introns for short, while the other regions are called exons exons. Researchers have discovered that signals for RNA splicing are short nucleotide sequences at the ends of introns. Particles called small nuclear ribonucleoproteins, or snRNPs (pronounced “snurps”), recognize these splice sites. snRNPs are located in the cell nucleus and are composed of RNA and protein molecules. The RNA in a snRNP particle is called a small nuclear RNA (snRNA); each molecule is about 150 nucleotides long. Several snRNPs join with additional proteins to form a spliceosome
5 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece) that cuts at specific points to release introns, then immediately joins together the two exons once adjacent to the intron. The idea of a catalytic role for snRNA arose from the discovery of ribozymes ribozymes, RNA molecules that function as enzymes.
RNA is often involved in catalyzing reactions. In a few cases, splicing can occur completely without proteins or even extra RNA molecules. For example, intron RNA in Tetrahymena splices itself.
The Functional and Evolutionary Importance of Introns
A number of genes are known to give rise to two or more different polypeptides, depending on which segments are considered exons during RNA processing: this is called alternative RNA splicing Split splicing. genes may also facilitate the evolution of new and potentially useful proteins. Proteins often have a modular architecture consisting of functional regions and structural regions called domains One omains. domain, for example, might include the active site, while another might attach the protein to a cellular membrane. Different exons often code for different domains of a protein. Introns increase the probability of potentially beneficial crossing over between genes. Introns increase the possibility that a crossover could switch one version of an exon for another version found on the homologous chromosome. THE SYNTHESIS OF PROTEINS Translation is the RNA-Directed Synthesis of a Polypeptide: A Closer Look tRNA), Transfer RNA (tRNA transfers amino acids from the cytoplasm to a tRNA ribosome. Each molecule of tRNA carries a specific amino acid, as determined by the anticodon which is part of the tRNA structure. The genetic message anticodon, will be translated as tRNA molecules deposite amino acids in the prescribed order and the ribosome joins the amino acids into a chain.
The Structure and Function of Transfer RNA
A tRNA molecule consists of a single RNA strand about 80 nucleotides long. There are about 45 types of tRNA, as some of them can recognize two or more different codons. The relaxation of base pairing rules that allows for multiple codons to code for the same amino acid is called wobble wobble.
A tRNA that binds to an mRNA codon specifying a particular amino acid must cary only that acid to the ribosome. Each amino acid joins to the correct tRNA by a specific enzyme called aminoacyl -tRNA aminoacylsynthetase. synthetase There are 20 of these enzymes in the cell, one enzyme for each amino acid. The synthetase catalyzes the reaction between tRNA and an amino acid through the hydrolysis of ATP. The resulting aminoacyl tRNA is called an activated amino acid.
6 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece)
A ribosome is made up of two subunits, constructed of proteins and RNA molecules named ribosomal (rRNA). RNA (rRNA) In eukaryotes, the sununits are constructed inside the nucleolus. Once large and small subunits attach to an mRNA molecule, they join to form a functional ribosome. Each ribosome has three binding sites for tRNA. The P site holds the tRNA carrying the growing polypeptide chain, while the A site holds the next amino acid to be added to the chain. Discharged tRNAs leave the ribosome from the E site.
Building a Polypeptide
Initiation. Initiation The initiation stage brings together mRNA, the first tRNA, and the two subunits of the ribosome. First, a small ribosomal subunit binds to both mRNA and the initiator tRNA, attaching to the leader segment at the 5’ end of the mRNA. The large ribosomal subunit will then attach to the small subunit. Proteins called initiation factors are required to bring it all together. The cell also spends energy in the form of a GTP molecule to form the initiation complex. Elongation. Elongation Amino acids are added one by one in a chian. The mRNA codon in the A site forms hydrogen bonds with the anticodon of an incoming tRNA (uses 2 molecules of GTP). The rRNA, functioning as a ribozymes, will catalyze the formation of a peptide bond that joins the polypeptide extending from the P site to the newly arrived amino acid. The ribosome now moves the tRNA in the A site to the P site. The tRNA that was previously in the P site is moved into the E site, where it leaves the ribosome. This move costs 1 GDP molecule. Termination. Termination Once a stop codon is reached, a protein called a release factorbinds directly to the stop codon in the A site, which causes a water molecule to be added to the polypeptide chain. This hydrolyzes the completed polypeptide from the tRNA in the P site.
Polyribosomes are strings of ribosomes that may trail along the same mRNA, which allow a cell to make many copies of a polypeptide quickly.
From Polypeptide to Functional Protein
7 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece) During and after synthesis, a polypeptide chain begins to coil and fold spontaneously, forming a functional protein of specific conformation. Additional steps, posttranslational modifications, may be required before the protein can begin functioning. Certain amino acids may be chemically modified, while enzymes may remove one or more amino acids. A single polypeptide chain can also be cleaved into two or more pieces. Signal Peptides Target Some Eukaryotic Peptides To Specific Destinations In the Cell Bound ribosomes are attached to the cytosolic side of rough endoplasmic reticulum. They work to make proteins of the endomembrane system, as well as proteins secreted from the cell. Synthesis of all proteins begins in the cytosol, when a free ribosomes starts to translate an mRNA molecule. The process will continue unless the growing polypeptide cues the ribosome to attach to the ER. The polypeptides of proteins destined for the endomembrane system or for secretion are marked by a signal peptide, peptide which targets the protein to the ER. The signal peptide will then be recognized as it emerges from the ribosome by a signal- recognition particle (SRP This particle functions as an adapter that ignalSRP). SRP brings the ribosome to a receptor protein built into the ER membrane. Point Mutations Can Affect Protein Structure and Function Mutations are changes in the genetic material of a cell (or virus). Point mutations are chemical changes in just one base pair of a gene. If a point mutation occurs in a gamete, it may be transmitted to offspring and to a succession of future generations. If the mutation has an adverse effect on the phenotype of a human or other animal, the mutant condition is referred to as a genetic disorder, or hereditary disease. The origins of sickle-cell disease can be traced to the mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin.
Types of Point Mutations
Substitutions. baseSubstitutions A base- pair substitution is the replacement of one nucleotide and its partner in the complementary DNA strand with another pair of nucleotides. Some substitutions are called silent mutations because they have no effect on the encoded protein. Other changes of a single nucleotide pair may switch an amino acid but have little effect on the protein. The new amino acid may have properties similar to the one it replaced, or it may e in a region of the protein where the exact sequence of amino acids is not essential to the proteins function.
8 UNIT THREE: GENETICS Chapter Seventeen: From Gene to Protein (Text from Biology, 6th Edition, by Campbell and Reece) However, base-pair substitutions in a crucial area such as the active site of an enzyme will significantly alter protein activity. Occasionally, such a mutation will lead to an improved protein or one with novel capabilities that enhance the success of the mutant organism and its descendants. But much more often, these mutations are detrimental. Substitution mutations are usually missense mutations mutations; that is, the altered codon will still code for an amino acid, but not necessarily the right one. If a point mutation changes a codon for an amino acid to a stop codon, it is known as mutations. nonsense mutations Insertions and Deletions. Insertions and deletions are additions or losses of nucleotide pairs in a gene. These mutations have a disastrous effect because the insertion or deletion may alter the triplet grouping of the genetic mutation. Such a mutation, called a f ram eshif t m utat io n, will occur whenever the number of nucleotides inserted or deleted is not a multiple of three. All the nucleotides downstream of the deletion/insertion will be improperly grouped.
Errors during DNA replication, repair, or recombination can lead to the mutations previously mentioned. Mutations resulting from such errors are called spontaneous mutations. A number of physical and chemical agents, called m utagen s, interact with DNA to cause mutations. In the 1920s, Hermann Muller discovered that fruit flies subjected to X-rays had increased genetic changes. Mutagenic radiation causes ultraviolet light that can produce disruptive thymine dimmers in DNA. Chemical mutagens fall into several categories. Base analogues are chemicals that are similar to normal DNA bases, but that pair incorrectly during DNA replication. Other chemical mutagens interfere with correct DNA replication by inserting themselves into the DNA and distorting the double helix. Other mutagens cause chemical changes to change their pairing properties. Researchers have been able to develop various methods to test the mutagenic activity of different chemicals. A major application is to test whether chemicals cause cancer. Since most carcinogens are mutagenic and most mutagens are carcinogenic, this approach makes sense.