Revised Protein Synthesis PDF

REVISED PROTEIN SYSNTHESIS  DNA, or deoxyribonucleic acid, contains the genes that determine who you are. This organic molecule control characteristics. DNA contains instructions for all the proteins the body makes. Proteins, in turn, determine the structure and function of all the cel...

REVISED PROTEIN SYSNTHESIS  DNA, or deoxyribonucleic acid, contains the genes that determine who you are. This organic molecule control characteristics. DNA contains instructions for all the proteins the body makes. Proteins, in turn, determine the structure and function of all the cells.  What determines a protein’s structure? It begins with the sequence of amino acids that make up the protein. Instructions for making proteins with the correct sequence of amino acids are encoded in DNA.  DNA is found in chromosomes. In eukaryotic cells, chromosomes always remain in the nucleus, but proteins are made at ribosomes in the cytoplasm or on the rough endoplasmic reticulum (RER). How do the instructions in DNA get to the site of protein synthesis outside the nucleus?  RNA is a small molecule that can squeeze through pores in the nuclear membrane. It carries the information from DNA in the nucleus to a ribosome in the cytoplasm and then helps assemble the protein. In short: DNA → RNA → Protein Discovering this sequence of events was a major milestone in molecular biology.  It is called the central dogma of biology. The two processes involved in the central dogma are transcription and translation. Another type of nucleic acid is responsible. This nucleic acid is RNA or ribonucleic acid.  Transcription This is the first part of the central dogma of molecular biology: DNA → RNA. Transcription is the mechanism by which the base sequence of a section of DNA representing a single gene is converted into the complementary base sequence of mRNA (copying base sequence).  Transcription may also be defined as the transfer of information from DNA to mRNA. It is the transfer of genetic instructions in DNA to mRNA. Transcription happens in the nucleus of the cell. During transcription, a strand of mRNA is made that is complementary to a strand of DNA called a gene.  A gene can easily be identified from the DNA sequence. A gene contains the basic three regions, promoter, coding sequence (reading frame), and terminator. Major parts of a gene  During protein synthesis, only one strand of DNA is used as a template for the formation of a complementary single strand of mRNA. Transcription starts when the DNA double helix unwinds when Hydrogen bonds that hold the two strands of DNA are broken down by the enzyme helicase. This exposes the nitrogenous bases in the DNA strands.  When the strands are separated, mRNA is then formed by linking of free nucleotides under the influence of RNA polymerase according to Chargiff’s rules (Adenine pairs with thymine or uracil to form two hydrogen bonds while cytosine pairs with guanine to form three hydrogen bonds). RNA Polymerase attaches to one DNA strand at a base sequence called promoter site.  Proteins called transcription factors, which have activators and repressor sequences that regulate gene expression are also attached to promoters. When enough mRNA is formed, the RNA Polymerase molecules leaves the DNA and the two strands reform the DNA double helix by closing the gap between the two DNA strands and reforming hydrogen bonds among the nitrogenous base.  This is done using an enzyme called ligase. When mRNA has been formed, it leaves the nucleus via the nuclear pores and carry the genetic code to the ribosomes in the cytoplasm.  Steps of Transcription Transcription takes place in three steps, called initiation, elongation, and termination. Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter.  This signals the DNA to unwind so the enzyme can “read” the bases in one of the DNA strands. The enzyme is ready to make a strand of mRNA with a complementary sequence of bases. The promoter is not part of the resulting mRNA. Elongation is the addition of nucleotides to the mRNA strand.  Termination is the ending of transcription. As RNA polymerase transcribes the terminator, it detaches from DNA. The mRNA strand is complete after this step.  Processing mRNA In eukaryotes, the new mRNA is not yet ready for translation. At this stage, it is called pre-mRNA, and it must go through more processing before it leaves the nucleus as mature mRNA. The processing may include the addition of a 5' cap, splicing, editing, and 3' polyadenylation (poly-A) tail.  These processes modify the mRNA in various ways. Such modifications allow a single gene to be used to make more than one protein. 5' cap protects mRNA in the cytoplasm and helps in the attachment of mRNA with the ribosome for translation. Splicing removes introns from the protein- coding sequence of mRNA.  Introns are regions that do not code for the protein. The remaining mRNA consists only of regions called exons that do code for the protein. Editing changes some of the nucleotides in mRNA. For example, a human protein called APOB, which helps transport lipids in the blood, has two different forms because of editing.  One form is smaller than the other because editing adds an earlier stop signal in mRNA. Polyadenylation adds a “tail” to the mRNA. The tail consists of a string of As (adenine bases). It signals the end of mRNA. It is also involved in exporting mRNA from the nucleus, and it protects mRNA from enzymes that might break it down.  Antisense /sense DNA The DNA sense strand looks like the messenger RNA (mRNA) transcript, and can therefore be used to read the expected codon sequence that will ultimately be used during translation (protein synthesis) to build an amino acid sequence and then a protein. For example, the sequence "ATG" within a DNA sense strand corresponds to an "AUG“ codon in the mRNA, which codes for the amino acid methionine.  However, the DNA sense strand itself is not used as the template for the mRNA; it is the DNA antisense strand which serves as the source for the protein code, because, with bases complementary to the DNA sense strand, it is used as a template for the mRNA.  Since transcription results in an RNA product complementary to the DNA template strand, the mRNA is complementary to the DNA antisense strand.  Hence, a base triplet 3′-TAC-5′ in the DNA antisense strand (complementary to the 5′-ATG-3′ of the DNA sense strand) is used as the template which results in a 5′-AUG-3′ base triplet in the mRNA. The DNA sense strand will have the triplet ATG, which looks similar to the mRNA triplet AUG but will not be used to make methionine because it will not be directly used to make mRNA.  The DNA sense strand is called a "sense" strand not because it will be used to make protein (it won't be), but because it has a sequence that corresponds directly to the RNA codon sequence. By this logic, the RNA transcript itself is sometimes described as "sense".  Translation This is the second part of the central dogma of molecular biology: RNA --> Protein. It is the process in which the genetic code in mRNA is read to make a protein. After mRNA leaves the nucleus, it moves to a ribosome, which consists of rRNA and proteins.  Translation happens on the ribosomes floating in the cytosol, or on the ribosomes attached to the rough endoplasmic reticulum. The ribosome reads the sequence of codons in mRNA, and molecules of tRNA bring amino acids to the ribosome in the correct sequence.  To understand the role of tRNA, you need to know more about its structure. Each tRNA molecule has an anticodon for the amino acid it carries. An anticodon is complementary to the codon for an amino acid. For example, the amino acid lysine has the codon AAG, so the anticodon is UUC.  Therefore, lysine would be carried by a tRNA molecule with the anticodon UUC. Wherever the codon AAG appears in mRNA, a UUC anticodon of tRNA temporarily binds. While bound to mRNA, tRNA gives up its amino acid.  With the help of rRNA, bonds form between the amino acids as they are brought one by one to the ribosome, creating a polypeptide chain. The chain of amino acids keeps growing until a stop codon is reached.  Ribosomes, which are just made out of rRNA (ribosomal RNA) and protein, have been classified as ribozymes because the rRNA has enzymatic activity. The rRNA is important for the peptidyl transferase activity that bonds amino acids. Ribosomes have two subunits of rRNA and protein.  The large subunit has three active sites called E, P, and A sites. These sites are important in the catalytic activity of ribosomes.  Protein Sysnthesis Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. In addition to the mRNA template, many other molecules contribute to the process of translation, such as ribosomes, tRNAs, and various enzymatic factors.  Translation Initiation: The small subunit binds to a site upstream (on the 5' side) of the start of the mRNA. It proceeds to scan the mRNA in the 5'-->3' direction until it encounters the START codon (AUG). The large subunit attaches and the initiator tRNA, which carries methionine (Met), binds to the P site on the ribosome.  Translation Elongation: The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs.  The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP. Briefly, the ribosomes interact with other RNA molecules to make chains of amino acids called polypeptide chains, due to the peptide bond that forms between individual amino acids.  Inside the ribosome, three sites participate in the translation process, the A, P, and E sites. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.  Translation Termination: Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released with the help of various releasing factors and the ribosome subunits dissociate and leave the mRNA.  After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.  At the initiation phase, the initiator tRNA carrying methionine with its anticodon encounters the AUG start codon at the P-site of the ribosome. During elongation, the ribosome translocates in the 5' to 3' direction of the mRNA, at which point the amino acids of tRNA in P-site and amino acid of tRNA in the A-site of the large subunit bond to each other via a peptide bond.  This repeated movement of the ribosome creates a long amino acid chain based on the codons in the mRNA. As the ribosome translocates, the tRNA leaves the ribosome through the E-site, while new tRNA enters the A-site in order to continue elongating the amino acid chain.  What Happens Next? After a polypeptide chain is synthesized, it may undergo additional processes. For example, it may assume a folded tertiary shape due to interactions among its amino acids. It may also bind with other polypeptides or with different types of molecules, such as lipids or carbohydrates.  Many proteins travel to the Golgi apparatus within the cytoplasm to be modified for the specific job they will do.