Understanding Gene Expression: Transcription and Translation, Slides of Biotechnology

Download Understanding Gene Expression: Transcription and Translation and more Slides Biotechnology in PDF only on Docsity! Chapter 17 From Gene to Protein Lecture Outline Overview • The information content of DNA is in the form of specific sequences of nucleotides along the DNA strands. • The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins. • Gene expression, the process by which DNA directs protein synthesis, includes two stages called transcription and translation. • Proteins are the links between genotype and phenotype. ° For example, Mendel’s dwarf pea plants lack a functioning copy of the gene that specifies the synthesis of a key protein, gibberellin. ° Gibberellins stimulate the normal elongation of stems. A. The Connection between Genes and Proteins 1. The study of metabolic defects provided evidence that genes specify proteins. • In 1909, Archibald Gerrod was the first to suggest that genes dictate phenotype through enzymes that catalyze specific chemical reactions in the cell. ° He suggested that the symptoms of an inherited disease reflect a person’s inability to synthesize a particular enzyme. ° He referred to such diseases as “inborn errors of metabolism.” • Gerrod speculated that alkaptonuria, a hereditary disease, was caused by the absence of an enzyme that breaks down a specific substrate, alkapton. ° Research conducted several decades later supported Gerrod’s hypothesis. • Progress in linking genes and enzymes rested on the growing understanding that cells synthesize and degrade most organic molecules in a series of steps, a metabolic pathway. • In the 1930s, George Beadle and Boris Ephrussi speculated that each mutation affecting eye color in Drosophila blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step. ° However, neither the chemical reactions nor the enzymes that catalyze them were known at the time. • Beadle and Edward Tatum were finally able to establish the link between genes and enzymes in their exploration of the metabolism of a bread mold, Neurospora crassa. ° They bombarded Neurospora with X-rays and screened the survivors for mutants that differed in their nutritional needs. IG Lecture Outlines 17-1 ° Wild-type Neurospora can grow on a minimal medium of agar, inorganic salts, glucose, and the vitamin biotin. • Beadle and Tatum identified mutants that could not survive on minimal medium, because they were unable to synthesize certain essential molecules from the minimal ingredients. ° However, most of these nutritional mutants can survive on a complete growth medium that includes all 20 amino acids and a few other nutrients. • One type of mutant required only the addition of arginine to the minimal growth medium. ° Beadle and Tatum concluded that this mutant was defective somewhere in the biochemical pathway that normally synthesizes arginine. ° They identified three classes of arginine-deficient mutants, each apparently lacking a key enzyme at a different step in the synthesis of arginine. ° They demonstrated this by growing these mutant strains in media that provided different intermediate molecules. ° Their results provided strong evidence for the one gene–one enzyme hypothesis. • Later research refined the one gene–one enzyme hypothesis. • First, not all proteins are enzymes. ° Keratin, the structural protein of hair, and insulin, a hormone, both are proteins and gene products. • This tweaked the hypothesis to one gene–one protein. • Later research demonstrated that many proteins are composed of several polypeptides, each of which has its own gene. • Therefore, Beadle and Tatum’s idea has been restated as the one gene–one polypeptide hypothesis. • Some genes code for RNA molecules that play important roles in cells although they are never translated into protein. 2. Transcription and translation are the two main processes linking gene to protein. • Genes provide the instructions for making specific proteins. • The bridge between DNA and protein synthesis is the nucleic acid RNA. • RNA is chemically similar to DNA, except that it contains ribose as its sugar and substitutes the nitrogenous base uracil for thymine. ° An RNA molecule almost always consists of a single strand. • In DNA or RNA, the four nucleotide monomers act like the letters of the alphabet to communicate information. • The specific sequence of hundreds or thousands of nucleotides in each gene carries the information for the primary structure of proteins, the linear order of the 20 possible amino acids. • To get from DNA, written in one chemical language, to protein, written in another, requires two major stages: transcription and translation. • During transcription, a DNA strand provides a template for the synthesis of a complementary RNA strand. IG Lecture Outlines 17-2 • The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals. • In laboratory experiments, genes can be transcribed and translated after they are transplanted from one species to another. ° This has permitted bacteria to be programmed to synthesize certain human proteins after insertion of the appropriate human genes. • Such applications are exciting developments in biotechnology. • Exceptions to the universality of the genetic code exist in certain unicellular eukaryotes and in the organelle genes of some species. ° Some prokaryotes can translate stop codons into one of two amino acids not found in most organisms. • The evolutionary significance of the near universality of the genetic code is clear. ° A language shared by all living things arose very early in the history of life—early enough to be present in the common ancestors of all modern organisms. • A shared genetic vocabulary is a reminder of the kinship that bonds all life on Earth. B. The Synthesis and Processing of RNA 1. Transcription is the DNA-directed synthesis of RNA: a closer look. • Messenger RNA, the carrier of information from DNA to the cell’s protein-synthesizing machinery, is transcribed from the template strand of a gene. • RNA polymerase separates the DNA strands at the appropriate point and bonds the RNA nucleotides as they base-pair along the DNA template. ° Like DNA polymerases, RNA polymerases can only assemble a polynucleotide in its 5’ 3’ direction. ° Unlike DNA polymerases, RNA polymerases are able to start a chain from scratch; they don’t need a primer. • Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends. ° RNA polymerase attaches and initiates transcription at the promoter. ° In prokaryotes, the sequence that signals the end of transcription is called the terminator. • Molecular biologists refer to the direction of transcription as “downstream” and the other direction as “upstream.” • The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. • Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules. • In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei. ° RNA polymerase II is used for mRNA synthesis. • Transcription can be separated into three stages: initiation, elongation, and termination of the RNA chain. • The presence of a promoter sequence determines which strand of the DNA helix is the template. ° Within the promoter is the starting point for the transcription of a gene. IG Lecture Outlines 17-5 ° The promoter also includes a binding site for RNA polymerase several dozen nucleotides “upstream” of the start point. • In prokaryotes, RNA polymerase can recognize and bind directly to the promoter region. • In eukaryotes, proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. • Only after certain transcription factors are attached to the promoter does RNA polymerase II bind to it. • The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex. ° A crucial promoter DNA sequence is called a TATA box. • RNA polymerase then starts transcription. • As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time. ° The enzyme adds nucleotides to the 3’ end of the growing strand. • Behind the point of RNA synthesis, the double helix re-forms and the RNA molecule peels away. ° Transcription progresses at a rate of 60 nucleotides per second in eukaryotes. • A single gene can be transcribed simultaneously by several RNA polymerases at a time. • A growing strand of RNA trails off from each polymerase. ° The length of each new strand reflects how far along the template the enzyme has traveled from the start point. • The congregation of many polymerase molecules simultaneously transcribing a single gene increases the amount of mRNA transcribed from it. • This helps the cell make the encoded protein in large amounts. • Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA. ° In prokaryotes, RNA polymerase stops transcription right at the end of the terminator. Both the RNA and DNA are then released. ° In eukaryotes, the pre-mRNA is cleaved from the growing RNA chain while RNA polymerase II continues to transcribe the DNA. Specifically, the polymerase transcribes a DNA sequence called the polyadenylation signal sequence that codes for a polyadenylation sequence (AAUAAA) in the pre- mRNA. At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme. The polymerase continues transcribing for hundreds of nucleotides. Transcription is terminated when the polymerase eventually falls off the DNA. 2. Eukaryotic cells modify RNA after transcription. • Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm. ° During RNA processing, both ends of the primary transcript are usually altered. ° Certain interior parts of the molecule are cut out and the remaining parts spliced together. IG Lecture Outlines 17-6 • At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap. • At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly-A tail. • These modifications share several important functions. ° They seem to facilitate the export of mRNA from the nucleus. ° They help protect mRNA from hydrolytic enzymes. ° They help the ribosomes attach to the 5’ end of the mRNA. • The most remarkable stage of RNA processing occurs during the removal of a large portion of the RNA molecule in a cut-and-paste job of RNA splicing. • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides. ° Noncoding segments of nucleotides called intervening regions, or introns, lie between coding regions. ° The final mRNA transcript includes coding regions, exons, which are translated into amino acid sequences, plus the leader and trailer sequences. • RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence. • This splicing is accomplished by a spliceosome. ° Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites. ° snRNPs are located in the cell nucleus and are composed of RNA and protein molecules. ° Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA). Each snRNA is about 150 nucleotides long. • The spliceosome interacts with certain sites along an intron, releasing the introns and joining together the two exons that flanked the introns. ° snRNAs appear to play a major role in catalytic processes, as well as spliceosome assembly and splice site recognition. • The idea of a catalytic role for snRNA arose from the discovery of ribozymes, RNA molecules that function as enzymes. ° In some organisms, splicing occurs without proteins or additional RNA molecules. ° The intron RNA functions as a ribozyme and catalyzes its own excision. ° For example, in the protozoan Tetrahymena, self-splicing occurs in the production of ribosomal RNA (rRNA), a component of the organism’s ribosomes. ° The pre-rRNA actually removes its own introns. • The discovery of ribozymes rendered obsolete the statement, “All biological catalysts are proteins.” • The fact that RNA is single-stranded plays an important role in allowing certain RNA molecules to function as ribozymes. • A region of the RNA molecule may base-pair with a complementary region elsewhere in the same molecule, thus giving the RNA a specific 3-D structure that is key to its ability to catalyze reactions. • Introns and RNA splicing appear to have several functions. IG Lecture Outlines 17-7 • Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules. ° The P site holds the tRNA carrying the growing polypeptide chain. ° The A site carries the tRNA with the next amino acid to be added to the chain. ° Discharged tRNAs leave the ribosome at the E (exit) site. • The ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the carboxyl end of the growing polypeptide. ° It then catalyzes the formation of the peptide bond. ° As the polypeptide becomes longer, it passes through an exit tunnel in the ribosome’s large unit and is released to the cytosol. • Recent advances in our understanding of the structure of the ribosome strongly support the hypothesis that rRNA, not protein, carries out the ribosome’s functions. ° RNA is the main constituent at the interphase between the two subunits and of the A and P sites. ° It is the catalyst for peptide bond formation. ° A ribosome can be regarded as one colossal ribozyme. • Translation can be divided into three stages: initiation, elongation, and termination. • All three phases require protein “factors” that aid in the translation process. • Both initiation and chain elongation require energy provided by the hydrolysis of GTP. • Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits. ° First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon. ° The small subunit then moves downstream along the mRNA until it reaches the start codon, AUG, which signals the start of translation. This establishes the reading frame for the mRNA. The initiator tRNA, already associated with the complex, then hydrogen-bonds with the start codon. ° Proteins called initiation factors bring in the large subunit so that the initiator tRNA occupies the P site. • Elongation involves the participation of several protein elongation factors, and consists of a series of three-step cycles as each amino acid is added to the proceeding one. ° During codon recognition, an elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresponding anticodon of tRNA carrying the appropriate amino acid. This step requires the hydrolysis of two GTP. ° During peptide bond formation, an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site. This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site. ° During translocation, the ribosome moves the tRNA with the attached polypeptide from the A site to the P site. Because the anticodon remains bonded to the mRNA codon, the mRNA moves along with it. The next codon is now available at the A site. IG Lecture Outlines 17-10 The tRNA that had been in the P site is moved to the E site and then leaves the ribosome. Translocation is fueled by the hydrolysis of GTP. Effectively, translocation ensures that the mRNA is “read” 5’ 3’ codon by codon. ° • The three steps of elongation continue to add amino acids codon by codon until the polypeptide chain is completed. • Termination occurs when one of the three stop codons reaches the A site. ° A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site. ° This frees the polypeptide, and the translation complex disassembles. • Typically a single mRNA is used to make many copies of a polypeptide simultaneously. ° Multiple ribosomes, polyribosomes, may trail along the same mRNA. ° Polyribosomes can be found in prokaryotic and eukaryotic cells. • A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide. • During and after synthesis, a polypeptide coils and folds to its three-dimensional shape spontaneously. ° The primary structure, the order of amino acids, determines the secondary and tertiary structure. • Chaperone proteins may aid correct folding. • In addition, proteins may require posttranslational modifications before doing their particular job. ° This may require additions such as sugars, lipids, or phosphate groups to amino acids. ° Enzymes may remove some amino acids or cleave whole polypeptide chains. ° Two or more polypeptides may join to form a protein. 2. Signal peptides target some eukaryotic polypeptides to specific destinations in the cell. • Two populations of ribosomes, free and bound, are active participants in protein synthesis. • Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol. • Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum. ° They synthesize proteins of the endomembrane system as well as proteins secreted from the cell. • While bound and free ribosomes are identical in structure, their location depends on the type of protein that they are synthesizing. • Translation in all ribosomes begins in the cytosol, but a polypeptide destined for the endomembrane system or for export has a specific signal peptide region at or near the leading end. ° This consists of a sequence of about 20 amino acids. • A signal recognition particle (SRP) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane. ° The SRP consists of a protein-RNA complex. IG Lecture Outlines 17-11 • After binding, the SRP leaves and protein synthesis resumes with the growing polypeptide snaking across the membrane into the cisternal space via a protein pore. ° An enzyme usually cleaves the signal polypeptide. • Secretory proteins are released entirely into the cisternal space, but membrane proteins remain partially embedded in the ER membrane. • Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system. ° In these cases, translation is completed in the cytosol before the polypeptide is imported into the organelle. ° While the mechanisms of translocation vary, each of these polypeptides has a “ZIP code” that ensures its delivery to the correct cellular location. • Prokaryotes also employ signal sequences to target proteins for secretion. 3. RNA plays multiple roles in the cell: a review. • The cellular machinery of protein synthesis and ER targeting is dominated by various kinds of RNA. ° In addition to mRNA, these include tRNA; rRNA; and in eukaryotes, snRNA and SRP RNA. ° A type of RNA called small nucleolar RNA (snoRNA) aids in processing pre-rRNA transcripts in the nucleolus, a process necessary for ribosome formation. ° Recent research has also revealed the presence of small, single-stranded and double- stranded RNA molecules that play important roles in regulating which genes get expressed. These types of RNA include small interfering RNA (siRNA) and microRNA (miRNA). ° The diverse functions of RNA are based, in part, on its ability to form hydrogen bonds with other nucleic acid molecules (DNA or RNA). ° It can also assume a specific three-dimensional shape by forming hydrogen bonds between bases in different parts of its polynucleotide chain. • DNA may be the genetic material of all living cells today, but RNA is much more versatile. • The diverse functions of RNA range from structural to informational to catalytic. 4. Comparing protein synthesis in prokaryotes and eukaryotes reveals key differences. • Although prokaryotes and eukaryotes carry out transcription and translation in very similar ways, they do have differences in cellular machinery and in details of the processes. ° Eukaryotic RNA polymerases differ from those of prokaryotes and require transcription factors. ° They differ in how transcription is terminated. ° Their ribosomes also are different. • One major difference is that prokaryotes can transcribe and translate the same gene simultaneously. ° The new protein quickly diffuses to its operating site. • In eukaryotes, the nuclear envelope segregates transcription from translation. ° In addition, extensive RNA processing is carried out between these processes. IG Lecture Outlines 17-12