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Unit 9 Notes: Transcription and Translation IB Biology Protein synthesis Introduction • Protein synthesis includes two reactions, transcription and translation. • The following table compares DNA and RNA DNA RNA Contains a 5-carbon sugar Contains a 5-carbon sugar 5-carbon sugar is deoxyribose 5-carbon sugar is ribose Each nucleotide has one of four nitrogenous bases Each nucleotide has one of four nitrogenous bases The nitrogenous bases are cytosine, guanine, adenine, and thymine The nitrogenous bases are cytosine, guanine, adenine, and uracil Double-stranded molecule Single-stranded molecule Transcription produces RNA molecules • The sections of DNA that code for polypeptides are called genes. • Genes are specific sequences of nitrogenous bases found in specific locations in a DNA molecule. • Molecules of DNA are found within the nucleus, proteins are synthesized outside the nucleus in the cytoplasm. Transcription produces RNA molecules • There has to be an intermediate molecule which carries the message of the DNA to the cytoplasm, where the enzymes, ribosomes, and amino acids are found. • The intermediate molecule is called messenger RNA or mRNA. Transcription produces RNA molecules • Transcription process: – The process of transcription begins when an area of DNA of one gene becomes unzipped. This process is very similar to the unzipping process in DNA replication. In this case only the area of the DNA where a gene is found is unzipped. – The two complementary strands of DNA are now single-stranded in the area of the gene. Transcription produces RNA molecules – RNA is a single stranded molecule which means that only one of the two strands of the DNA will be used as a template to create the mRNA molecule. The enzyme RNA polymerase is used as the catalyst for this process. – As RNA polymerase moves along the strand of DNA acting as the template, RNA nucleotides float into place by complementary base pairing. – Base pairing is the same as DNA replication with the exception that adenine pairs with uracil. Transcription produces RNA molecules • The following pertains to transcription: – Only one of the two strands of DNA is copied, the other strand is not used. – mRNA is always single stranded and shorter than the DNA that is copied from as it is a complementary copy of only one gene. The central dogma • The idea that information passes from genes on DNA to an RNA copy, the RNA copy then directs the production of proteins at the ribosome by controlling the sequence of amino acids is called the central dogma. • The central dogma can be summarized as follows: • DNA → RNA → Proteins Transcription: DNA → RNA • Transcription has some similarities to replication. • First, the double helix must be opened to expose the base sequence of the nucleotides. Helicase unzips the DNA in replication, however in transcription RNA polymerase separates the two DNA strands. • The RNA polymerase also allows polymerization of RNA nucleotides as base-pairing occurs along the DNA template. • The RNA polymerase must first combine with a region of the DNA strand called a promoter. RNA polymerase only allows assembly in the 5’ to 3’ direction. Transcription: DNA → RNA • Which strand of DNA is copied: – One strand is complementary to the other, so there would be a difference in the code of the strands. – The genetic code is made up of codons which are three nucleotide triplets. – The codons are specific for certain amino acids. Therefore, complementary strands mean different codons, different amino acids, and different proteins. Transcription: DNA → RNA • The DNA strand that carries the genetic code is called the sense strand (or coding strand). The other strand is called the antisense strand (or the template strand). • The sense strand has the same sequence as the newly transcribed RNA except thymine is in the place of uracil in the DNA strand. • The antisense strand is the strand that is copied during transcription. • The promoter region for a particular gene determines which DNA strand is the antisense strand. For any particular gene, the promoter is always on the same DNA strand. Transcription: DNA → RNA • The promoter region is a short sequence of bases that is not transcribed. • Once RNA polymerase has attached to the promoter region for a particular gene transcription begins. • The DNA opens and a transcription bubble occurs. This bubble contains the antisense DNA strand, the RNA polymerase, and the growing RNA transcript. Transcription: DNA → RNA • The terminator – The sections of DNA involved in transcription are: promoter → transcription unit → terminator. The transcription bubble moves from the DNA promoter region towards the terminator. – The terminator is a sequence of nucleotides that, when transcribed, causes the RNA polymerase to detach from the DNA, which stops transcription. Transcription: DNA → RNA • The transcript carries the code of the DNA and is referred to as messenger RNA (mRNA). • In eukaryotes, transcription continues beyond the terminator for a significant number of nucleotides until it is eventually released from the DNA molecule. Transcription: DNA → RNA • Nucleoside triphosphates (NTP’s) containing three phosphates and the 5-carbon sugar, ribose are paired with the appropriate exposed bases of the antisense strand. • Polymerization of the mRNA strand occurs with the catalytic help of RNA polymerase and the energy provided from the release of two phosphates from NTP. • This portion of the transcription process is referred to as elongation. Transcription: DNA → RNA • Post-transcription processing – Eukaryotic DNA contains large stretches of noncoding DNA, which is different from prokaryotic DNA. – The large stretches of non-coding DNA are called introns. – In order to make the mRNA strand functional in eukaryotes, the introns must be removed. – Prokaryotic mRNA does not require processing because no introns are present. The genetic code is written in triplets • Polypeptides are composed of amino acids covalently bonded together in a specific sequence. • The message written into the mRNA molecule is the message that determines the order of the amino acids. • Every three bases of RNA codes for one of the 20 amino acids. • Any set of three bases is called a triplet, in an RNA molecule the triplets are called codons. Translation results in the production of a polypeptide • There are three types of RNA molecules. – mRNA (messenger RNA) – each mRNA is a complementary copy of DNA that codes for a single polypeptide. – tRNA (transfer RNA) – each type of tRNA transfers one of the 20 amino acids to the ribosome for polypeptide formation. – rRNA (ribosomal RNA) – each ribosome is composed of rRNA and ribosomal protein. Translation results in the production of a polypeptide • Each tRNA molecule contains an anticodon that determines which of the 20 amino acids will be transferred to mRNA. • Translation process: – The mRNA will locate a ribosome and align with it so that the first two codon triplets are within the boundaries of the ribosome. – A specific tRNA molecule then floats in – its tRNA anticodon must be complementary to the first codon triplet of the mRNA molecule, thus the first amino acid is brought in. Translation results in the production of a polypeptide • While the first tRNA ‘sits’ in the ribosome holding the amino acid, a second tRNA floats in and brings a second amino acid. • The second tRNA matches its three anticodon bases with the second codon triplet of the mRNA. • An enzyme now catalyzes a condensation reaction between the two amino acids and the resulting covalent bond between them is called a peptide bond. • The next step in the translation process involves the breaking of the bond between the first tRNA molecule and the amino acid that is transferred in. Translation results in the production of a polypeptide • The bond is no longer needed as the second tRNA is currently bonded to its own amino acid which is covalently bonded to the first amino acid. • The first tRNA floats away into the cytoplasm to bond with another amino acid of the same type. • The ribosome that has only one tRNA in it now moves one codon triplet down the mRNA molecule, which puts the second tRNA in the ribosome position that the first originally occupied and creates room for a third tRNA to float in with its amino acid. Translation results in the production of a polypeptide • This process repeats itself as another peptide bond forms, the ribosome moves down the mRNA by another triplet each time until the last codon triplet is reached. • The final codon triplet will be a triplet that does not act as a code for an amino acid, it signals a ‘stop’ to the translation process. • The entire polypeptide breaks away from the final tRNA molecule and becomes a free floating polypeptide in the cytoplasm. The one gene/one polypeptide hypothesis • In the early 1940s, experimental work was performed which led to the hypothesis that every one gene of DNa produced one enzyme. Which was soon amended to include all proteins. • It was later discovered that many proteins are actually composed of more than one polypeptide and it was proposed that each polypeptide required a separate gene. • Researchers in the last few years have discovered that at least some genes are not that straightforward. One gene may lead to a single mRNA molecule, but the mRNA molecule may then be modified in many different ways, which may result in the production of a different polypeptide. Ribosomes • Translation occurs within a ribosome. • Ribosomes can be seen with an electron microscope, each consists of a large subunit and a small subunit. • The subunits are composed of ribosomal RNA (rRNA) molecules and many distinct proteins. • rRNA proteins are generally small and are associated with the core of the RNA subunits. Roughly two-thirds of the ribosome mass is rRNA. Ribosomes • The molecules of the ribosomes are constructed in the nucleolus of eukaryotic cells and exit the nucleus through the membrane pores. • Prokaryotic ribosomes are smaller than eukaryotic ribosomes. They are also molecularly different than eukaryotic ribosomes. • Decoding of a strand of mRNA to produce a polypeptide occurs in the space between the two subunits. Ribosomes • In this area, there are binding sites for mRNA and three sites for binding tRNA, as shown below: Site Function A site Holds the tRNA carrying the next amino acid to be added to the polypeptide chain P site Holds the tRNA carrying the growing polypeptide chain Site from which tRNA that has lost its amino acid is discharged E site Ribosomes • Polypeptide chains are assembled in the cavity between the two subunits. • This area is generally free of proteins, so binding of mRNA and tRNA is carried out by rRNA. • tRNA moves sequentially through the three binding sites: from the A site, to the P site, and finally to the E site. • The growing polypeptide chain exits the ribosome through a tunnel in the large subunit core. Translation: RNA → protein • The translation process involves the following phases: – Initiation – Elongation – Translocation – Termination Translation: RNA → protein • Codons carry the genetic code from DNA to the ribosomes by mRNA. • There are 64 possible codons, three codons have no complementary tRNA anticodon (these are stop codons). • There is a start codon (AUG) that signals the beginning of a polypeptide chain. • This codon also encodes the amino acid methionine. Translation: RNA → protein • The Initiation Phase – The start codon (AUG) is on the 5’ end of all mRNAs. – Each codon, other than the three stop codons, attach to a particular tRNA. The tRNA has a 5’ end and a 3’ end like all other nucleic acid strands. – The 3’ end of the tRNA is free and has the base sequence CCA. This is the site of amino acid attachment. Translation: RNA → protein • Because there are complementary bases in a single stranded tRNA, hydrogen bonds form in four areas. This causes the tRNA to fold and take on a three dimensional structure. • If the molecule is flattened, it has the twodimensional appearance of a clover leaf. • One of the loops of the clover leaf contains an exposed anticodon. This anticodon is unique to each type of tRNA. It is the anticodon that pairs with a specific codon of mRNA. Translation: RNA → protein • Each of the 20 amino acids binds to the appropriate tRNA due to the action of an enzyme, each of the 20 amino acids has its own enzyme. • The active site of each enzyme allows it to only fit between a specific amino acid and the specific tRNA. The actual attachment of the amino acid and tRNA requires energy that is supplied by ATP. • At this point the structure is called an activated amino acid, and the tRNA may now deliver the amino acid to a ribosome to produce the polypeptide chain. Translation: RNA → protein • So, the first step in initiation of translation is when an activated amino acid – methionine attached to a tRNA with the anticodon UAC – combines with an mRNA strand and a small ribosomal subunit. • The small subunit moves down the mRNA until it contacts the start codon (AUG). This contact starts the translation process. Translation: RNA → protein • Hydrogen bonds form between the initiator tRNA and the start codon. • Next, a large ribosomal subunit combines with these parts to form the translation initiation complex. • Joining the initiation complex are proteins called initiation factors that require energy from guanosine triphosphate (GTP) for attachment. GTP is an energy-rich compound very similar to ATP. Translation: RNA → protein • The elongation phase – Once initiation is complete elongation occurs. – This phase involves tRNAs bringing amino acids to the mRNA-ribosomal complex in the order specified by the codons of mRNA. – Proteins called elongation factors assist in binding to the tRNAs to the exposed mRNA codons at the A site. The initiator tRNA then moves the the P site. – The ribosomes catalyze the formation of peptide bonds between adjacent amino acids Translation: RNA → protein • The translocation phase – The translocation phase actually happens during the elongation phase. – Translocation involves the movement of tRNAs from one site of the mRNA to another. – First, a tRNA binds with the A site. Its amino acid is then added to the growing polypeptide chain by a peptide bond. Translation: RNA → protein • This causes the polypeptide chain to be attached to the tRNA at the A site. • The tRNA then moves to the P site. It transfers its polypeptide chain to the new tRNA that moves into the now exposed A site. • The now empty tRNA is transferred to the E site where it is released. This process occurs in the 5’ to 3’ direction. • Therefore, the ribosomal complex is moving along the mRNA toward the 3’ end. Translation: RNA → protein • The termination phase – The termination phase begins when one of the three stop codons appears at the open A site. – A protein called a release factor then fills the A site. The release factor does not carry an amino acid. – It catalyzes hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain. Translation: RNA → protein • This frees the polypeptide, releasing it from the ribosome. • The ribosome then separates from the mRNA and splits into its two subunits. • The termination phase completes the process of translation. • Proteins synthesized in this manner have several different destinations. • If they are produced by free ribosomes, the proteins are primarily used by the cell, if they are produced by ribosomes bound to the ER, they are primarily secreted or used in lysosomes. Proteins • Protein functions and structures – The following are just a few proteins and their functions: Protein Function Hemoglobin Protein containing iron that transports oxygen from the lungs to all parts of the body in vertebrates Actin and myosin Proteins that interact to bring about muscle movement (contraction) in animals Insulin A hormone secreted by the pancreas that aids in maintaining blood glucose level in vertebrates Immunoglobulins Group of proteins that act as antibodies to fight bacteria and viruses Digestive enzyme that catalyzes the hydrolysis of starch Amylase Proteins • There are proteins that perform structural tasks, that store amino acids, and some that have receptor functions so cells can respond to stimuli. • There are four levels of organization in protein structure (primary, secondary, tertiary, and quaternary): Proteins • Primary organization: – The primary level of protein structure is the unique sequence of amino acids held together by peptide bonds in each protein. – There are 20 amino acids and they can be arranged in any order. – The order or sequence in which the amino acids are arranged is determined by the nucleotide base sequence in the DNA of the organism. Proteins • Every organism has its own DNA, so every organism has its own unique proteins. • The primary structure is simply the amino acid chain attached by peptide bonds. • Polypeptide chains may include hundreds of amino acids. Proteins • Changing one amino acid in a chain may completely alter the structure and function of the protein. • For example, sickle cell disease is caused by just one amino acid change in the normal hemoglobin protein of red blood cells. The result is that red blood cells are unable to carry oxygen. Proteins • Secondary organization: – Hydrogen bonds form between the oxygen from the carboxyl group of on amino acid and the hydrogen from the amino group of another – Secondary structure does not involve the side chains, or R groups of amino acids. The two most common configurations of secondary structure are the α-helix and the β-pleated sheet. – Both have regular repeating patterns. Proteins • Tertiary organization: – The polypeptide chain bends and folds over itself because of interactions among R groups and the peptide backbone, which results in a definite three-dimensional conformation. – Interactions that cause tertiary organization include: • Covalent bonds between sulfur atoms to create disulfide bonds – these are often called bridges because they are strong. Proteins • Hydrogen bonds between polar side chains. • Van der Waals interactions among hydrophobic side chains of the amino acids. – these interactions are strong because many hydrophobic side chains are forced inwards when the hydrophilic side chains interact with water toward the outside of the molecule. • Ionic bonds between positively and negatively charged side chains. Proteins • Tertiary structure is extremely important in determining the specificity of proteins such as enzymes. Proteins • Quaternary organization: – Quaternary structure is unique in that it involves multiple polypeptide chains which combine to form a single structure. – Not all proteins have quaternary structure. – All the bonds mentioned in the previous three levels of organization are involved in this level. Proteins • Some proteins with quaternary structure include prosthetic or non-polypeptide groups, these are called conjugated proteins. • Hemoglobin is an example of a conjugated protein, because it contains four polypeptide chains, each of which contains a heme group (non-polypeptide). Heme contains iron which binds to oxygen. Proteins • Fibrous and globular protein – Fibrous proteins are composed of many polypeptide chains in a long narrow shape, they are usually insoluble in water. • One example is collagen, which is important in the structure of connective tissue of humans. • Actin is another example, which is important in human muscle contraction. Proteins • Globular proteins are more three-dimensional in shape and are mostly soluble in water. – Hemoglobin is one type of globular protein. – The hormone insulin is another globular protein, which regulates blood glucose levels in humans. Proteins • Polar and non-polar amino acids – Amino acids are often grouped according to the properties of their R groups. – Amino acids with non-polar side chains are hydrophobic. Non-polar amino acids are found in regions of proteins that are linked to the hydrophobic are of the cell membrane. Proteins • Polar amino acids have hydrophilic properties, and are found in regions of proteins that are exposed to water. • Membrane proteins include polar amino acids toward the interior and exterior of the membrane, these amino acids create hydrophilic channels through which polar substances can move. Proteins • Polar and non-polar amino acids are important in determining the specificity of an enzyme. • Each enzyme has an active site, in which only a specific substrate can bind. • Combination is possible through ‘fitting’ which involves the general shapes and the polarity of the substrate and the amino acids at the active site.