Lecture 8: Transcription Notes on Molecular Genetics PDF

Notes on Molecular Genetics Lecture#8 Transcription - From DNA to RNA The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into...

Notes on Molecular Genetics Lecture#8 Transcription - From DNA to RNA The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name transcription. Figure 46 Gene expression levels of Gene A and B Transcription Produces RNA Complementary to One Strand of DNA All of the RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication. Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then 1  Notes on Molecular Genetics acts as a template for the synthesis of an RNA molecule. As in DNA replication, the nucleotide sequence of the RNA chain is determined by the complementary base-pairing between incoming nucleotides and the DNA template. When a good match is made, the incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction. The RNA chain produced by transcription—the transcript—is therefore elongated one nucleotide at a time, and it has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template. Transcription, however, differs from DNA replication in several crucial ways: - Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. - In addition, because they are copied from only a limited region of the DNA, RNA molecules are much shorter than DNA molecules. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter. The enzymes that perform transcription are called RNA polymerases. Like the DNA polymerase that catalyzes DNA replication, RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5′-to-3′ 2  Notes on Molecular Genetics direction Figure 47. The substrates are nucleoside triphosphates (ATP, CTP, UTP, and GTP); as for DNA replication, a hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward. Figure 47: RNA polymerase binding DNA for transcription The almost immediate release of the RNA strand from the DNA as it is synthesized means that many RNA copies can be made from the same gene in a relatively short time, the synthesis of additional RNA molecules being started before the first RNA is completed. When RNA polymerase molecules follow hard on each other's heels in this way, each moving at about 20 nucleotides per second (the speed in eucaryotes), over a thousand transcripts can be synthesized in an hour from a single gene. Although RNA polymerase catalyzes essentially the same chemical reaction as DNA polymerase, there are some important differences between the two enzymes: First, and most obvious, RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. 3  Notes on Molecular Genetics Second, unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference may exist because transcription need not be as accurate as DNA replication. Unlike DNA, RNA does not permanently store genetic information in cells. RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides), and the consequences of an error in RNA transcription are much less significant than that in DNA replication. Although RNA polymerases are not nearly as accurate as the DNA polymerases that replicate DNA, they nonetheless have a modest proofreading mechanism. If the incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that mimics the reverse of the polymerization reaction, except that water instead of pyrophosphate is used. RNA polymerase hovers around a misincorporated ribonucleotide longer than it does for a correct addition, causing excision to be favored for incorrect nucleotides. However, RNA polymerase also excises many correct bases as part of the cost for improved accuracy. COMPARE BETWEEN DNA POLYMERASE AND RNA POLYMERAZES 4  Notes on Molecular Genetics Cells Produce Several Types of RNA Table 6: Principal Types of RNAs Produced in Cells TYPE FUNCTION OF RNA mRNAs messenger RNAs, code for proteins rRNAs ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis tRNAs transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids snRNAs small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA snoRNAs small nucleolar RNAs, used to process and chemically modify rRNAs microRNA microRNA is the name of a family of molecules that helps cells control the kinds and amounts of proteins they make. Other function in diverse cellular processes, including telomere synthesis, noncoding X-chromosome inactivation, and the transport of proteins into the ER RNAs Each transcribed segment of DNA is called a transcription unit. In eucaryotes, a transcription unit typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). In bacteria, a set of adjacent genes is often transcribed as a unit; the resulting mRNA molecule therefore carries the information for several distinct proteins. Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The way in which RNA polymerases 5  Notes on Molecular Genetics perform these tasks differs somewhat between bacteria and eucaryotes. Because the process in bacteria is simpler, we look there first. The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. Bacterial RNA polymerase is a multisubunit complex. A detachable subunit, called sigma (σ) factor, is largely responsible for its ability to read the signals in the DNA that tell it where to begin transcribing (Figure 48). RNA polymerase molecules adhere only weakly to the bacterial DNA when they collide with it, and a polymerase molecule typically slides rapidly along the long DNA molecule until it dissociates again. However, when the polymerase slides into a region on the DNA double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, it binds tightly to it. The polymerase, using its σ factor, recognizes this DNA sequence by making specific contacts with the portions of the bases that are exposed on the outside of the helix. 6  Notes on Molecular Genetics Figure 48: Steps of transcription in bacteria After the RNA polymerase binds tightly to the promoter DNA in this way, it opens up the double helix to expose a short stretch of nucleotides on each strand. Unlike a DNA helicase reaction , this limited opening of the helix does not require the energy of ATP hydrolysis. Instead, the polymerase and DNA both undergo reversible structural changes that result in a more energetically favorable state. With the DNA unwound, one of the two exposed DNA strands acts as a template for complementary base-pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain. After the first ten or so nucleotides of RNA have been synthesized (a relatively inefficient process during which polymerase synthesizes and discards 7  Notes on Molecular Genetics short nucleotide oligomers), the σ factor relaxes its tight hold on the polymerase and evenutally dissociates from it. During this process, the polymerase undergoes additional structural changes that enable it to move forward rapidly, transcribing without the σ factor (Step 4 infigure 48). Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal in the DNA, the terminator (described below), where the polymerase halts and releases both the DNA template and the newly made RNA chain. After the polymerase has been released at a terminator, it reassociates with a free σ factor and searches for a new promoter, where it can begin the process of transcription again. How do the signals in the DNA (termination signals) stop the elongating polymerase? For most bacterial genes a termination signal consists of a string of A-T nucleotide pairs preceded by a two-fold symmetric DNA sequence, which, when transcribed into RNA, folds into a “hairpin” structure through Watson-Crick base-pairing. As the polymerase transcribes across a terminator, the hairpin may help to wedge open the movable flap on the RNA polymerase and release the RNA transcript from the exit tunnel. At the same time, the DNA-RNA hybrid in the active site, which is held together predominantly by U-A base pairs (which are less stable than G-C base pairs because they form two rather than three hydrogen bonds per base pair), is not sufficiently strong enough to hold the RNA in place, and it dissociates causing the release of the polymerase from the DNA, perhaps by forcing open its jaws. Thus, in some respects, transcription termination seems to involve a reversal of the structural transitions that happen during initiation. The process of termination also is an example of a common theme in this chapter: the ability of RNA to fold into specific structures figures prominantly in many aspects of decoding the genome. 8  Notes on Molecular Genetics Promoter sequences are asymmetric (Figure 43), and this feature has important consequences for their arrangement in genomes. Since DNA is double-stranded, two different RNA molecules could in principle be transcribed from any gene, using each of the two DNA strands as a template. However a gene typically has only a single promoter, and because the nucleotide sequences of bacterial (as well as eucaryotic) promoters are asymmetric the polymerase can bind in only one orientation. The polymerase thus has no option but to transcribe the one DNA strand, since it can synthesize RNA only in the 5′ to 3′ direction. The choice of template strand for each gene is therefore determined by the location and orientation of the promoter. Genome sequences reveal that the DNA strand used as the template for RNA synthesis varies from gene to gene (Figure 49). Figure 49: Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Approximately 0.2% (9000 base pairs) of the E. coli chromosome is depicted here. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template. 9