mRNA Synthesis Lecture Notes

○ ○ ○ When RNA is translated to a protein it usually means gene expression’s molecules go from being information storage molecules to metabolic and structural functional molecules. If there is an error during the transcription of a gene, The final protein might not be functional. If there is an error during translation of a gene, the protein might not be functional. Transcription mRNA Synthesis ○ The segment of the DNA molecule where messenger RNA synthesis begins is called the promoter region. ○ The complementary messenger RNA strand that would be synthesized from the DNA base sequence of 5'-CTGAC-3' would be 3'-GACUG-5' ○ The structure that causes the synthesis of RNA to cease is known as the transcription terminator. ○ What is a difference between DNA replication and RNA transcription?In DNA replication the helix is never closed after it is opened by helicase, but once RNA polymerase has finished transcribing a region, the double helix closes. ○ How do you know that the gene being transcribed in the animation is in a prokaryote? ○ The initiation of transcription involves a sigma factor. Are both strands of DNA used in making RNA? DNA is a two stranded molecule. Only one strand is used to transcribe mRNA. During transcription the two strands separate and RNA polymerase synthesizes mRNA based on the sequence of one the two strands. Then the two DNA strands join together again. Either strand may be used to produce mRNA. One strand does not exclusively code for mRNA while the other does not. But for any given mRNA strand only one of the DNA strands is used.The complementary nature of DNA would make it very unlikely that both strands could code simultaneously for functional proteins. Here is an analogy with language. Imagine that each letter in the alphabet had a complimentary letter. For example A might match with Q, B with X, and so on. If we were to write a normal English word the complementary sequence would be determined by the letters in that word and could not be changed. It is very unlikely that the complementary sequence would be a recognizable word. ○ The enzyme that accomplishes transcription is termed ○ The enzyme that accomplishes transcription is termed RNA polymerase. ○ The transcription enzyme first attaches to the promoter of the gene. ○ RNA polymerase is able to open the DNA double helix as it moves down the template. What type of enzymatic activity does this mean RNA polymerase must possess? Helicase activity. ○ Which of the following statements about a structural gene is TRUE? Transcription of structural genes requires DNA sequences that are not part of the gene itself. ○ Why are nucleotide triphosphates used to polymerize RNA? The triphosphate is a high energy molecule so that polymerization is spontaneous. How is mRNA transcribed without disrupting a DNA molecule? Just like DNA synthesis, mRNA synthesis is done along small sections of DNA that are uncoiled and the two strands separated. Once the mRNA is created from one of the DNA strands, that portion of the DNA rejoins and recoils as more DNA opens up further down. In this way a continuous strand of mRNA can be synthesized without uncoiling a long sequence of DNA and separating the two strands. Only separating a small portion of the DNA at a time reduces the probability of damage to the molecule. ○ ○ The Rho protein is involved in the termination stage of transcription. ○ Sigma factor recognizes the promoter region. ○ Formation of a holoenzyme occurs first in transcription. ○ If the sigma factor didn’t bind to the RNA polymerase, the RNA polymerase would not bind to the template DNA. ○ If the rho protein did not bind to the RNA, the RNA polymerase would not dissociate from the RNA and the template DNA. ○ Bicyclomycin is an antibiotic that is effective against many gram negative bacteria. It works by inhibiting Rho. What direct effect would this have on a bacteria? It would prevent termination of RNA transcription, causing mRNA transcripts to be abnormally long. Transcription Factors What is meant by gene activation? In a multicellular organism every cell contains every gene found in the organism. Not all genes are actually used in every cell. The specialization of cells into different types means that some genes are used in some cells and not in others. Also many genes are only used at certain times. How are genes turned on and off? There are many different mechanisms for controlling transcription of mRNA. If a gene is ‘turned on' then transcription proceeds followed by translation and production of a protein. If a gene is ‘turned off' then transcription is halted. Different kinds of cells have different patterns of transcription (known as gene expression). Gene expression also varies within a cell depending on the influence of external factors. ○ ○ ○ The basal transcription factors are essential for transcription and they cannot increase the rate of transcription by themselves. The assembly of transcription factors begins upstream from the transcription start site. A large region of a promoter has been deleted upstream of a gene. Basal transcription factors won’t bind to the template DNA, therefore RNA polymerase will not bind to the template DNA and transcription will not occur. RNA Modification in Eukaryotes. RNA Splicing What property of RNA and DNA allows for the cutting of the molecule and removing of pieces without destroying the entire structure? Both RNA and DNA are modular structures. They consist of repeated elements: sugars and bases. Each repeated element has a 3' and a 5 ‘ end. These repeated elements link to one another in a chain, attaching the 3' end of one molecule to the 5' end of another molecule. If a section of the chain is removed the two separated sections will have exposed 3' and 5' ends. Thus the two separated sections can easily attach to one another and form a complete chain. The modular nature of DNA and RNA allow the molecules to be modified relatively easily when necessary. ○ Splicing joins together two exons. ○ ○ ○ ○ ○ The snRNPs are also called snurps. RNA splicing proper sequenced by U2 binds to branch site, then lariat is formed, finally 3’ splice site is cut. 5' ...CCGAGGUACGAUUUGAGUCGGCUACAGUAUUGA.... 3' 5' ...CCGAGGUACGAUUUGAGUCGGCUACAGUAUUGA.... 3' Introns are generally considered “free to mutate,” meaning that if a mutation occurs within the intron, it will not affect the functional protein. An exception to this axion is If there is a mutation in the 3' or 5' splice site, the snurp won't recognize it, and it will not be removed from the mRNA. Processing of Gene Information ○ Eukaryotic mRNA is when a cap is added to their 5’ end, a poly-A tail is added to their 3’ end, and each usually specifies only a single protein. ○ Splicing joins together joins exons. ○ Eukaryotes require a 5’ cap and a poly-A tail but prokaryotes don’t need to transport their RNA out of the nucleus, so they don’t need these features. ○ Scientists should remove the introns to a eukaryotic gene before they insert it into a bacterial chromosome to be expressed. Translation and the Genetic Code ○ ○ ○ ○ ○ Translation is the synthesis of proteins from mRNA. The tRNA that was in the P site moves into the E site as the ribosomes shift down the mRNa by a distance of three nucleotides. Translation is terminated when a stop codon is presented at the A site. A mutation occurred in the third codon position of a gene, but the protein still functions normally. Due to the genetic code being redundant, mutations in the third position of the codon often result in the same amino acid being specified. RNA synthesis called “transcription” and protein synthesis called translation because RNA synthesis makes a complementary copy of the DNA using nucleic acid and protein synthesis requires the information in the RNA to be charged into a different type of molecule. The Stages of Translation Protein Synthesis 2 ○ New amino acids (other than the initial f-Met) enter at A-site. ○ At the E-site, transfer RNA is released. ○ The unusual thing about the initiator tRNA in prokaryotes is that it carries a modified amino acid AND it does not bind to the ribosome at the A site. ○ The formation of a bond between the peptide in the P site and the amino acid in the A site stimulates the ribosome to move down one codon. ○ If an aminoacyl-tRNA synthetase added the wrong amino acid to a tRNA, the tRNA would carry the wrong amino acid, and it would be incorporated into the growing protein. ○ The initiating transfer RNa, carrying formylmethionine, binds to P site. ○ The amino acids carried by the transfer RNA are joined by peptide bonds. ○ If an aminoacyl-tRNA synthetase added the wrong amino acid to a tRNA,The tRNA would carry the wrong amino acid, and it would be incorporated into the growing protein. Translation Initiation What is the function of ribosomal RNA? Ribosomes are complex structures composed of both protein and RNA. About two thirds of the mass of a ribosome is RNA, making ribosomal RNA the most abundant type of RNA in the cell. Ribosomal RNA functions in binding mRNA and tRNA and then moving them precisely through the ribosome in order to add amino acids to a polypeptide chain. The small subunit of a ribosome contains a sequence of ribosomal RNA that binds mRNA at the beginning of translation. Without this binding site translation might not begin at the appropriate location on the mRNA. ○ Initiation of translation includes mRNA, ribosomal subunits, and initiator tRNA. ○ The Shine-Dalgarno sequence is found on the mRNA. ○ During the initiation of translation, GTP is used to facilitate the binding of the mRNA, and the initiator tRNA to the ribosome. ○ All nascent proteins begin with methionine. ○ If the Shine-Dalgarno sequence was mutated, then the mRNA would not bind to the small ribosomal subunit in the proper location. Translation Elongation Why are ribosomes needed for protein synthesis? Ribosomes are the central structures of protein synthesis. They bring all the components needed to build a protein together in a controlled fashion that causes amino acids to link up in a chain. Ribosomes have sites that bind mRNA and tRNA and move them through the ribosome. The movement of RNA through the ribosome adds the amino acids to the chain, one at a time. Without ribosomes, the tRNA and the mRNA would have to spontaneously come together. After the association of the two kinds of RNA, the amino acids attached to the tRNA would have to link together. Without a ribosome this would take a very long time. Ribosomes are the organizers of protein synthesis. ○ ○ ○ ○ ○ During translation elongation, amino acids are brought to the ribosome with the help of EF-Tu. The tRNA that was in the P site moves into the E site is a result of translocation. Peptidyl transferase catalyzes the formation of peptide bonds and it is present in the 50S ribosomal subunits. Ribosomes recognize the proper tRNA to bind by only a tRNA with an anticodon that is complementary to the covod will bind in the A-site. GTP used in Elongation aids in the binding of the incoming tRNA to the A site of the ribosome AND It aids in translocating the ribosome one codon down the mRNA. Translation Termination Why does mRNA need a stop codon? When a ribosome reaches the codon UAG, UAA, or UGA on an mRNA strand then protein synthesis is terminated. A protein known as a release factor binds to the ribosome and adds a water molecule to the end of the amino acid chain. This reaction hydrolyzes the polypeptide chain from the tRNA, allowing the protein to exit the ribosome. Without the stop codon, protein synthesis would simply stop without releasing the completed protein. ○ ○ ○ ○ ○ Termination is terminated when a stop codon is presented at the A site. The protein that promotes translation termination is called release factor. No tRNA can recognize a stop codon and it is recognized by a release factor protein. A single point mutation has occurred in a gene causing a stop codon to appear early in the translation of the protein. This will affect the protein by causing termination to occur earlier. Then, the protein will be abnormally short. Free carboxylic acid group is formed during termination