Chapter 17: From Gene to protein A gene is a region of DNA that can be expressed to produce a final functional product

that is either a polypeptide or an RNA molecule. One gene-one polypeptide hypothesis 1. George Beadle and Edward Tatum based this idea on their study of Neurospora crassa, a red bread mold. Wild type strain only needed the minimal medium to grow(contains sucrose,essential minerals; inorganic salts and 1 vitamin. 2. The mold uses a multistep pathway to synthesise the amino acid arginine from the precursor. 3. Beadle and Tatum identified 3 mutants unable to synthesise arg. for 3 different reasons. Each mutant had a metabolic block at a different step in the pathway. Eg. Class II mutants failed to grow on the minimal medium/ minimal medium supplied with ornithine. Adding either citruline or arginine allowed these mutants to grow. Deduction: Class II mutants lacked the enzyme converting ornithine citruline. Adding citruline to the medium bypasses the metabolic block, allowing the mold to survive. The other class of mutants lacked different enzymes. Conclusion: The various mutations were abnormal variations of different genes, each gene dictationg the production of 1 enzyme; 1 gene-1 enzyme hypothesis. Restated to 1 gene- 1 polypeptide hypothesis ( proteins that are not enzymes are nevertheless gene products. Not only that, but many proteins are constructed from 2 or more different polypeptide chains, and thus 2 genes code for this protein) Transcription and Translation Transcription the synthesis of RNA by DNA (DNA mRNA)

DNA(template for assembling complementary sequence of RNA) Translation- synthesis of polypeptides under the direction of mRNA. Bacterial Cell Eukaryotic cell No nucleus, mRNA immediately Nuclear envelope separates transcription from translation. translated without additional pathway Nucleus: transcription, Cytoplasm: translation. Original RNA transcript(pre-mRNA) processed in various ways before leaving nucleus as mRNA.

Codons: Triplets of bases For each gene, 1 RNA strand functions as a template for transcription. The base pairing rule for DNA synthesis also guides transcription. But Uracil takes place of thymine. During translation, an mRNA read as a sequence of base triplets called codons. Each codon specifies an amino acid to be added to the polypeptide chain.. mRNA is read from the 5 to the 3 direction.

Transcription is the DNA-directed synthesis of RNA Molecular components of transcription 1. mRNA is transcribed from the template strand of a gene. RNA polymerase pries the DNA strand apart and joins the RNA nucleotides as they base-pair along the DNA template. 2. RNA polymerase can only assemble its nucleotide in its 5 to 3 direction(like DNA polymerase) However, unlike DNA polymerase, RNA polymerase is able to start a chain from scratch without a primer!! 3.Promoter- DNA sequence where RNA polymerase attaches itself and initiates transcription. Terminator- Sequence that signals the end of transcription(in bacteria) Transcription Unit: Stretch of DNA transcribed into an RNA molecule. Synthesis of an RNA transcript (3 stages; initiation, elongation and termination) RNA Polymerase Binding and Initiation of Transcription 1. The promoter of a gene includes within it the transcription start point (nucleotide where RNA synthesis begins) and extends upstream from the start point. 2.Promoter: binding site for RNA polymerase, determining where transcription starts, determines which of the 2 DNA strands is used as the template. 3. In bacteria, RNA polymerase itself recognizes and binds to the promoter. In eukaryotes, transcription factors(a protein collection) mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase II bind to it. 4. Transcription initiation complex = the whole complex of transcription factors and RNA polymerase II bound to the promoter(in a eukaryotic promoter, a TATA box a crucial DNA promoter is also needed together with the transcription factors in forming the initiation complex. 5. Once the polymerase is attached firmly to the promoter DNA, the 2 DNA strands unwind, the enzyme initiates RNA synthesis at the start point on the template strand/ starts transcribing the template strand. Elongation of the RNA strand 1. As RNA polymerase moves along the DNA, it continues to untwist the double helixz(exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides. 2. Nucleotides are added by the enzyme from the 3 end of the growing RNA molecule as it continues along the double helix. 3. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript from the 5 to the 3 direction. DNA strands reform a double helix. Termination of Transcription 1. Eventually the RNA transcript is released, and the polymerase detaches from the DNA. 2. Termination=differ in bacteria and eukaryote. 3. In bacteria, transcription proceeds through a terminator sequence in the DNA. The transcribed terminator functions as the termination signal, causing the polymerase to detach from the DNA and release the transcript, which is available for immediate use as mRNA. 4. In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence, which codes for a polyadenylation signal(AAUAAA) in the pre mRNA. Then,at a point about 10 to 35 nucleotides downstream from the signal, proteins associated with the growing RNA transcript cut it free from the polymerase, releasing the pre-mRNA .Pre-mRNA then undergoes processing.

Eukaryotic cells modify RNA after transcription Pre-mRNA is modified in specific ways before being dispatched to the cytoplasm. During this RNA processing, both ends of the primary transcript are altered. Certain sections are cut off and the remaining parts are spliced together. The modified mRNA is then ready for translation. Alteration of mRNA ends How does alteration of the 5 and 3 ends of the pre-mRNA affect the mRNA exiting the nucleus? The 5 cap and poly-a tail facilitate mRNA export from the nucleus, prevent the mRNA from being degraded by hydrolytic enzymes, and facilitate ribosome attachment. 1. Each end of a pre-mRNA is modified in a particular way ; the 5 end being synthesised first, receiving a 5 cap (a modified form of a guanine nucleotide is added to onto the 5 end) after transcription of the first 20-40 nucleotides. 2. The3 end of the pre-mRNA is also modified before exiting the nucleus. At this end, an enzyme adds 50-250 more adenine(A) molecules, forming a poly-A tail. 3. Function of the 5 end and poly-A tail: Facilitate the export of mature mRNA from the nucleus, protect mRNA from degradation by hydrolytic enzymes, help ribosomes attach to the 5 and 3 ends of the mRNA once the mRNA reaches the cytoplasm. 4.UTRs (untranslated regions) will not be translated into proteins but play roles in ribosome binding. Split Genes and RNA splicing 1.RNA splicing is the removal of large portions of RNA molecules that is initially synthesized (similar to editing a video) 2. Most eukaryotic genes and their RNA transcripts have long non-coding stretches of nucleotides, regions that are not translated. The non-coding segments of nucleic acid that lie between coding regions are known as introns. The other regions are called exons, because they are eventually expressed, usually, by being translated into amino acid sequences.(Exceptions include UTRs of the exons at the ends of the RNA, which make up part of the mRNA but are not translated into the protein). (think of exons as sequences of RNA that exit the nucleus.. The terms introns anf exons are used for both RNA and DNA sequences that encode them.) 3. In making a 10 transcript from agene, RNA polymerase II transcribes both introns and exons from the DNA, but the mRNA molecule that enters the cytoplasm is an abridged version.The introns are cut out from the molecule and the exons spliced together, forming an mRNA molecule with a continuous coding sequence. This is the process of RNA splicing. 4. Pre-mRNA splicing i) Small nuclear ribonucleoproteins(snRNPs) and other proteins form a molecular complex called a spliceosome on a pre-mRNA molecule containing introns and exons. ii) Within the spliceosome, snRNA(small nuclear RNA; RNA in a snRNP particle) base- pairs with nucleotides at specific sites along the intron. iii) The spliceosome cuts the pre-mRNA, releasing the intron, and at the same time splices the exon together. The spliceosome then comes apart, releasing mRNA, which now contains only exons. Ribozymes 1.In some organisms, RNA splicing can occur without proteins or even additional molecules: The intron RNA functions as a ribozyme and catalyses its own excision!! EG> 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 idea that all biological catalysts are proteins. 2. Three properties of RNA that enable some RNA molecules to function as enzymes: i) RNA is single stranded-a region of an RNA molecule may base-pair with a complementary region elsewhere in the same molecule,giving the molecule a particular 3D structure.(a specific structure is essential to the catalytic function of proteins, just as it is for enzymatic proteins. ii) Some of the bases in RNA contain functional groups that may participate in catalysis (like certain amino acids in an enzymatic protein.) iii) The ability of RNA to H-bond with other nucleic acid molecules (DNA/RNA) adds specificity to its catalytic activity. (eg. complementary base-pairing between the RNA of a spliceosome and the RNA of a primary RNA transcript precisely locates the region where the ribozyme catalyses splicing.) The Functional and Evolutionary Importance of Introns. 1. The presence of introns enable more than 1 kind of polypeptide to be encoded by a single gene. Many genes are known to give rise to two or more different types of polypeptides, depending on which segments are treated as exons during RNA processing, this is known as alternative RNA splicing.(eg. sex differences in the fruit fly is largely due to the differences in how the male and female splice the RNA transcribed from certain genes) 2.Results from the Human Genome Project suggest that alternative RNA splicing is 1 reason humans can get along with a relatively small number of genes. Because of alternative splicing, the no. of different protein products an organism produces can be much greater than its number of genes. 3. Proteins often have a modular architecture consisting of discrete structural and functional regions called domains. One domain of an enzymatic protein, for instance, might include the active site, while another may attach the protein to a cellular membrane. In quite a few cases, different exons code for the different domains of a protein. 4. The presence of introns in a gene may facilitate the evolution of new and potentially useful proteins as a result of exon shuffling. Introns increase the probability of potentially beneficial crossing over between the exons (of alleles)-simply by providing more places for crossing over without interrupting coding sequences. This could lead to new proteins with novel combination of functions. Occasionally a beneficial variant might rise from the shuffling. Translation is the RNA-directed Synthesis of a Polypeptide. Molecular Components of Translation 1. As a molecule of mRNA is moved through a ribosome, codons are translated one-by-one into amino acids. 2. The interpreters are tRNA molecules, each type with a specific anticodon at one end and a corresponding amino acid at the other end. 3. A tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon H-bonds to a complementary codon on the mRNA. 4. Ribosomes help facilitate this coupling with binding sites for tRNA and mRNA. The Structure and Function of Transfer RNA; tRNA 1. Transfer RNA are transcribed from DNA templates. In an eukaryotic cell, tRNA is made in the nucleus and must travel to the cytoplasm, where translation occurs. 2. In both bacterial and eukaryotic cell, each tRNA molecule is used repeatedly, picking up its designated amino acid in the cytosol, depositing them onto a polypeptide chain at the ribosome, then leaves the ribosome, ready to pick up another amino acid. 3. A tRNA molecule consists of a single tRNA strand(only about 80 nucleotides long, compared to hundreds of nucleotides for most mRNA molecules. 4. The presence of complementary stretches of bases that can H-bond to each other enables the tRNA molecule to fold back upon itself and form a molecule with a 3-D structure.

5. A tRNA molecule looks like a clover leaf.(20 structure). 6. It twists and folds into a compact 3-D structure that is roughly L-shaped.The loop extending from one end of the L includes the anticodon, the particular base-triplet that base-pairs to the mRNA codon. From the other end of the L-shaped tRNA molecule protrudes its 3 end , which is the attachment site for an amino acid. 30 structure- (all tRNAs have a similar L-shaped 3D structure that allows them to fit into the P and A sites of the ribosome). The cloverleaf structure becomes the 3D L-shaped structure through coaxial stacking of the helices, which is a common RNA tertiary structure motif. Ribosomes 1. Ribosome facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis. 2. A ribosome is made up of a large and small subunits. These ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNAs or rRNAs. 3. In addition to a binding site for mRNA, each ribosome has 3 binding sites for tRNA, the P site, A site and E site. 4. The P site (peptidyl-site) holds the tRNA carrying the growing polypeptide chain, while the A site(aminoacyl-tRNA site) holds the tRNA carrying the next amino-acid to be added to the chain. Discharged tRNAs leave the ribosome from the E-site (exit site). 5. 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. 6. As the polypeptide becomes longer, it passes through an exit tunnel in the ribosome s large subunit. When the polypeptide is complete, it is released to the cytosol through the exit tunnel. Building a polypeptide chain 1.Translation can be divided into 3 stages: Initiation, elongation and termination 2.For certain aspects of initiation and elongation, energy is required (provided by the hydrolysis of GTP- guanosine triphosphate, a molecule closely related to ATP) Ribosome association and Initiation of Translation 1.The initiation stage of translation brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the 2 subunits of a ribosome. 2. A small ribosomal unit binds to a mRNA molecule. In a bacterial cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence the mRNA just upstream the start codon. An initiator of tRNA, with the anticodon UAC base-pairs with the start codon AUG(Met-methionine) 3. The arrival of a large ribosomal subunit completes the initiation complex. (Transcription initiation complex= union of mRNA, initiator tRNA, and a small ribosomal subunit followed by the attachment of a large ribosomal subunit) 4.Proteins called initiation factors are required to bring all the translation components together. GTP provides energy for the assembly. Upon completion of the initiation, the initiator tRNA is in the P site and the vacant A site is ready for the next aminoacyl tRNA. Note: A polypeptide is always synthesized in one direction, from the initial methionine at one end (N-terminus) toward the final amino acid at the carboxyl end (C-terminus) Elongation of the polypeptide chain In the elongation stage of translation, amino acids are added one-by-one to the preceding amino acid. Each addition involves the participation of several proteins called elongation factors and occurs in a 3-step cycle.

1. Codon recognition: The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. 2. Peptide-bond formation: An rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P-site. (This step removes the polypeptide from the tRNA in the P site and attaches it to the amino acid on the tRNA in the A site. 3. Translocation: The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNA s, bringing the next codon to be translated into the A site. Termination of translation Elongation continues until a stop codon in the mRNA reaches the A site of the ribosome. Stop codon ( signal to stop translation): UAG, UAA, UGA 1. When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a release factor, a protein shaped like a tRNA, instead of an aminoacyl tRNA. 2. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain The polypeptide is thus freed from the ribosome. 3. The 2 ribosomal subunits and the other components of the assembly dissociates with the hydrolysis of 2 more GTP molecules. Polyribosomes An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Polyribosomes are found in both bacterial and eukaryotic cells. They enable a cell to make many copies of a polypeptide very quickly. Completing and targeting the functional protein The process of translation is often not sufficient to make a functional protein. Therefore, polypeptide chains undergo modifications after the translation process as well as some of the mechanisms used to target completed proteins to specific sites in the cell. Protein Folding and Post-Translational Modifications 1. During its synthesis, a polypeptide chain begins to coil & fold spontaneously due to its 10 amino acid sequence, forming a protein with a specific shape; a 3D molecule with 20 & 30 structure. 2. Thus, a gene determines 10 structure and the 10 structure in turn determines shape. In most cases, a chaperone protein(chaperonin) helps the polypeptide chain fold correctly. 3. Post-translational modifications (additional steps) may be required before the protein can begin its particular job in the cell; a) certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. b) Enzymes may remove 1 or more amino acids from the leading (amino) end of the polypeptide chain. c) In some cases, a polypeptide chain may be enzymatically cleaved into 2 or more pieces. (e.g. the protein insulin is first synthesized as a polypeptide chain but is activated only after an enzyme cuts out a central part of the chain, leaving a protein made up of 2 polypeptide chain connected by disulphide bridges). d) In other cases, 2 or more polypeptides that are synthesized separately may come together, becoming the subunits of a protein that has a quarternary structure(40), e.g Hemoglobin.

Targeting polypeptides to specific locations In eukaryotic cells active in protein synthesis, there are 2 types of ribosome that are evident; free and bound ribosomes. Both are identical and can switch from being free to bound. What determines whether a ribosome will be free in the cytosol or bound to the rough ER at any given time? Protein synthesis always begin in the cytosol until completion-unless the growing polypeptide chain cues the ribosome to attach to the ER. A signal peptide marks protein polypeptides destined for the endomembrane system or for secretion, which targets the protein to the ER. The signal peptide is recognised as it emerges from the ribosome by a protein RNA complex called SRP. The signal mechanism for targeting proteins to the ER 1. Polypeptide synthesis begins on a free ribosome in the cytosol when the free ribosome starts to translate an mRNA molecule. 2. An SRP (signal-recognition particle) binds to the signal peptide, halting synthesis momentarily. 3. The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. 4. The SRP leaves, and polypeptide synthesis resumes, with simultaneous translocation across the membrane. ( The signal peptide stays attached to the translocation complex.) 5. The signal cleaving enzyme cuts off the peptide. 6. The rest of the completed polypeptide leaves the ribosome and folds into its final conformation. 7. The protein is further processed in the ER and then in the Golgi. Finally, a transport vesicle will convey it to the plasma membrane for release from the cell. Note: other kinds of signal peptides are used to target polypeptides to other organelles that are not part of the endomembrane system. However, translation is completed in the cytosol before the polypeptide is imported into the organelle. Point Mutation can Affect Protein Structure and Function Mutations are responsible for the huge diversity of genes found among organisms because they are the ultimate source of new genes. Point mutation is the change in the DNA nucleotide sequence of a particular region of a chromosome. It refers to the chemical changes in a single base-pair of a gene. Types of Point Mutation Base-pair substitution, insertion & deletion A. Substitutions A base-pair substitution is the replacement of 1 nucleotide and its partner with another pair of DNA nucleotides. 1. Silent mutation Change in a base-pair does not affect the encoded protein as the base-pair still codes for the same amino acid . This is due to the redundancy of the genetic code. 2. Missense Mutation a) A substitution that changes 1 amino acid into another b) May have little effect on the protein : i) Has similar properties to that of the amino acid it replaces, or, ii) Located in a protein region where the exact sequence of amino acid is not important to the protein s function. c) However, when the alteration of single amino acid occurs in a crucial area of a protein, there will be a major change in the protein. Eg. Sickle- cell anemia d) Substitution mutations are usually missense mutations in which the altered codon still codes for an amino acid and thus makes sense, although not necessarily the right sense

3. Nonsense mutation a) Base-pair substitution in the DNA nucleotide sequence which result in a premature stop codon. A completely non-functional protein is produced. B. Insertions and Deletions 1. The additions or losses of nucleotide pairs in a gene. Have a disastrous effect on the resulting protein more often than substitutions do. 2. May alter the reading frame of the genetic message, the triplet grouping of bases on the mRNA read during translation.(known as frameshift mutation) 3. Frameshift mutation will occur whenever the no. of nucleotides being inserted or deleted is not a multiple of 3. (All the nucleotides that are downstream of the deletion or insertion will be improperly grouped into codons, hence resulting in an extreme missense, usually ending sooner or later in nonsense and premature termination.) 4. Unless the frameshift mutation is very near the end of the gene, the protein is almost certainly non-functional. Mutagens 1. A no. of physical and chemical agents, called mutagens interact with DNA in ways that cause mutations. 2.Mutagenic radiation, a physical mutagen, includes ultraviolet (UV) light, which can cause disruptive thymine dimers in DNA. 3. Chemical mutagens fall into several categories: Base analogs are chemicals that are similar to normal DNA bases but that pair incorrectly during DNA replication. Some other chemical mutagens interfere with the correct DNA replication by inserting themselves into the DNA and distorting the double helix. Still other mutagens cause chemical changes in bases that change their pairing properties. While gene expression differs among the domains of life, the concept of a gene is universal. Although bacterial and eukaryotic cells carry out transcription and translation in very similar ways, there are certain differences in cellular machinery and in details of the processes in these 2 domains. Like bacteria, archaea are prokaryotes. However, archaea share many aspects of the mechanism of gene expression with eukaryotes, as well as bacteria. Comparing Gene Expression in Bacteria, Archaea and Eukarya. RNA polymerase Transcription factors Termination of transcription Translation Bacteria and RNA polymerase differ from each other. The single RNA polymerase of archaea resembles the 3 eukaryotic RNA polymerases Both archaea and eukaryotes use a complex set of transcription factors, unlike bacteria. Different in eukaryotes and bacteria. Archaeal transcription termination may be more like the eukaryotic process. Bacterial and eukaryotic ribosomes are slightly different. Archaeal ribosomes are the same size as bacterial ribosome but their sensitivity to chemical inhibitors most likely match that of eukaryotic ribosomes. Slightly different in bacteria and eukaryotes, Archaeal process is more like that of bacteria. Bacteria Eukarya Archaea Absent Present ( nuclear * similar to bacteria envelope) Complicated mechanisms for targeting proteins to organelles in eukaryotic cells

Initiation of translation Compartmental organization Mechanisms