Transcription of DNA Assessment Structure 7.3.1 State that transcription is carried out in a 5’ – 3’ direction 7.3.2 Distinguish between the sense and antisense strands of DNA 7.3.3 Explain the process of transcription in prokaryotes, including the role of the promoter region, RNA polymerase, nucleoside triphosphates and the terminator 7.3.4 State that eukaryotic RNA needs the removal of introns to form mature mRNA DNA does contain codes for everything in your body but, DNA doesn’t leave the nucleus. In fact, the nucleus acts to protect the DNA, and it is only unprotected when mitosis occurs. In order to get the information from DNA out to the rest of the cell and form the structures of proteins and enzymes needed, we need RNA, or ribonucleic acid. RNA is similar to DNA, except it normally exists as a single strand, and it has ribose as its sugar. Another major difference is that in RNA, thymine is replaced by a new nitrogen base, called uracil. There are several different forms of RNA: mRNA or messenger RNA, tRNA or transfer RNA, and rRNA or ribosomal RNA. The central process can be summerized as follows: DNA RNA Protein How do DNA and RNA work together? The job of RNA is to take a copy of the information on a strand of DNA, and to take this info out into the cell. This process is called transcription. The new strand of RNA is transcribed in the 5’ – 3’ direction, and is carried out by RNA polymerase. The next question is, “how do DNA and RNA know what information to use?” In order to understand how this process works, it is much easier to study this in prokaryotes than eukaryotes – and this study was carried out in 1961 by Jacob and Monod. They studied E. coli and how it produced lactose digesting enzymes. They found that E. coli does not produce lactose digesting enzymes when grown on a medium without lactose, but the enzyme is present within minutes of lactose being added to the medium. Jacob and Monod discovered that genes can be “switched on” or “switched off” as needed, simply by transcribing or not transcribing them into mRNA. When a gene is transcribed, the mRNA ends up being translated into a protein. The model for switching genes on and off is called the operon model. An operon includes the promoter, the operator and the structural gene. To transcribe the structural gene for the lactose digesting enzyme into the required mRNA, the RNA polymerase needs to attach to the promoter section of the DNA so that it can make the primer. However, the operator section is situated next to the promoter section – this is a binding site for a repressor protein produced by another gene (called a regulator gene) elsewhere on the bacterial DNA. If the repressor protein is not present, it will attach to the operator site and prevent the RNA polymerase from binding to the gene – stopping transcription. However, if lactose is present, it will bind to the repressor protein, changing its shape, making it unable to bond to the operator. Without the repressor, the DNA can be transcribed into mRNA, and the lactose enzyme will be made. What this all means is that in order for the enzyme to be made, the DNA has to be read. The idea of it only being made when needed, supports the idea that it is not DNA, but something else. What would be the result if enzymes and proteins were made directly from DNA? Transcription As we saw above, the act of transcription is similar to the replication of DNA. It goes in a 5’ – 3’ direction, but helicase is not used to unwind the DNA. Also, since only one strand is copied, the complementary strands have a difference in codes. As you recall, the genetic code is made up of codons, or pieces of three base pairs. The codons are specific for certain amino acids or punctuation signals. Therefore, complementary strands mean different codons, different amino acids and different proteins. Since only 1 strand of the double helix is transcribed, there are special terms for each - The strand being transcribed is called the anti-sense strand. - The strand not being used is the sense strand. - Thus, the mRNA is complimentary to the anti-sense strand and is a RNA copy of the sense strand In Eukaryotic cells a promoter region for a particular gene determines which DNA strand is the antisense strand. The promoter is always on the same DNA strand. The promoter is a short sequence of bases that allows RNA polymerase to bond to it. Once RNA polyerase has attached to the promoter region, for a particular gene, the process begins. The DNA opens and a transcription bubble occurs. Ribonucleoside triphosphates bond using hydrogen bonds and base pairing. RNA polymerase then attaches the individual nucleotides together into a single strand of RNA. When the rNTP’s are attached together with the help of RNA polymerase, energy is provided by the release of two phosphates. This part of transcription is referred to as elongation. The transcription bubble moves from the promoter region towards the terminator. The terminator is a sequence of nucleotides that, when transcribed, causes the RNA polymerase to detach from the DNA. Transcription stops and the RNA transcript is detached from the DNA. It is now referred to as messenger RNA or mRNA. Prokaryotes v. Eukaryotes There are differences in how RNA works in eukaryotes. In prokaryotes, genes occur in uninterrupted sections of DNA, so no further processing of RNA is required. There are no non-coding portions. In eukaryotes, many genes have coding sections of DNA that are interrupted by long, non-coding sequences (repetitive sequences) RNA polymerase transcribes the entire section of DNA, but before RNA leaves the nucleus, a cap is added to the 5’ end (protects RNA from phosphates and nucleases and assists in protein synthesis). A tail is added to the 3’ end, which increases the stability of the mRNA. Enzymes then precisely cut the bonds between the coding sequences (called exons) and the non-coding sequences (called introns) – this process is called splicing, and once complete the mRNA is ready to go to the cytoplasm. This is often referred to as mature mRNA. EXTRA - The bad side of Transcription Using RNA to get the message out from DNA is good, but this system can be abused! A small group of viruses, called retroviruses, cause their host cells to produce a special enzyme called reverse transcriptase. The virus has RNA as its genetic material – but it can also be used to make proteins directly. Reverse transcriptase is used to transcribe viral RNA into a single strand of DNA, and a double helix between DNA and RNA is formed. The enzyme then digests the RNA strand, and the enzyme is used again to produce a complimentary strand of DNA, and thus a double helix strand of DNA. This then becomes part of the host cell’s DNA. The host is then made to transcribe many RNA copies of this material, and to produce many protein coats so that many new viruses can be assembled. Cancer causing viruses and HIV are examples We have discovered a way to make use of the reverse transcriptase enzyme! If we have the mRNA needed for a certain gene, we can use reverse transcriptase to produce the DNA coding for that gene, without all the extra information that was cut out (introns). This new, smaller gene is much easier to work with and can be inserted into the DNA of an organism. An example of this is the production of insulin. Insulin is a protein – thus comes from mRNA. Using reverse transcriptase, the DNA for the gene was created, then was inserted into E. coli bacteria. The bacteria began producing human insulin (because it was based on human DNA) and makes life better for those who cannot produce this protein themselves! More on this in Biotechnology.