Transcription and Translation: Part 2 of 3: Transcription Tsonwin Hai, Ph.D. Professor Department of Molecular and Cellular Biochemistry The Ohio State University College of Medicine

Study Objective At the end of this module, you should be able to: Understand gene structure, a few terms and concepts—in a broad sense—and be able to apply them. Identify the features of transcription from a list of multiple choices. Predict transcription product. Answer the practice exam questions at the end. Textbook: Principles of Medical Biochemistry, Meisenberg and Simmons, Mosby, Inc. copyright 1998; Chapter 8, The human genome and its expression (particularly pp )

Section I: A Few Terms Cis-acting element, Trans-acting factor: Promoter, Enhancer, Silencer Promoter is a segment of DNA that specifies the binding of RNA polymerase and controls the transcription of a specific gene. The promoter functions like a “switch” to turn on or off the genes and can be positively or negatively regulated. Enhancer is a segment of DNA that can greatly increase the transcription of a gene in the following manner: (a) independent of its distance from the transcriptional start (enhancer can be thousands or tens of thousands bases pair away from the transcription start site (TSS), (b) independent of its orientation, and (c) independent of its location (enhancer can be downstream, upstream, or even in the midst of the gene). Silencer is opposite to enhancer: Instead of increasing transcription, it decreases it. A cis-acting element refers to a short stretch of DNA sequence that constitutes a signal for specific protein(s) to bind. Protein factor that bind to the cis-acting elements is called the trans-acting factor. RNA Polymerases: RNA polymerases are enzymes that synthesizes RNA using DNA as template. In prokaryotes, the same RNA polymerase makes mRNA, tRNA and rRNA. In contrast, in eukaryotes, different polymerases make these RNA molecules: Polymerase I (Pol I), Pol II, and Pol III. Click the “Attachments” (on the upper right corner) to see specific polymerases in eukaryotes.

Section I: A Few Terms (Cont’d) General transcription factor, Sequence-specific transcription factor, Transcription initiation complex: General transcription factors are required for the transcription of all promoters. They include RNA polymerase and other protein factors (such as TBP, TAFs); these general transcription factors form a large complex called the initiation complex or the basal transcription machinery. This complex binds to the DNA around the transcriptional start site. In the absence of any upstream sequence-specific transcription factors, the initiation complex gives rise to a basal, low level of transcription. Sequence-specific transcription factors bind to specific cis-acting elements (that is, specific DNA sequences). They communicate (interact) with the basal transcription machinery either directly or indirectly to up- or down-regulate transcription. Below are schematics of direct (left) and indirect (right, through co-factors) interactions. The dotted line indicates the machinery and the arrow indicates the start of transcription. Click the “Attachments” (on the upper right corner) to see more description of the transcription initiation complex (for Polymerase II).

Section II: Transcription Reaction The initiation complex binds to DNA around the transcriptional start site, unwinds the DNA so that the incoming ribonucleotide triphosphates can form Watson-Crick base pairing with the template DNA. The complex then slides along the DNA and adds new nucleotides (ribonucleotide not deoxyribonucleotide) to the growing RNA chain, with the double stranded DNA in front of the complex unwinding (green arrow) and DNA behind the complex rewinding (red arrow). When the complex encounters the “termination signals,” it stops transcription and falls off the DNA. Several websites have animated images for transcription: RNA Molecule

Section III: Features of Transcription de novo synthesis: The 5' end of a new RNA chain starts with pppG or pppA, no primer is required. This is in contrast to DNA replication, which requires primer. Lower fidelity (than DNA replication): Although mistakes are corrected during RNA synthesis, the fidelity of transcription is much lower than that of DNA replication. 5’ to 3’ synthesis: Although double stranded DNA is required to function as a promoter, only one of the two DNA strands is used as the template and the RNA molecule is synthesized from 5’ to 3’. That is, the first incoming nucleotide is the 5’ most nucleotide of the resulting RNA molecule. The question below is designed to help you understand this point.

If the transcriptional machinery travels along the double stranded DNA below at the direction from your left to your right, which of the following RNA will be produced? (A) 5’-AGGCUUACGCCA-3’ (B) 5’-TGGCGTAAGCCT-3’ (C) 5’-AGGCTTACGCCA-3’ (D) 5’-UGGCGUAAGCCU-3’ (E) 5’-ACCGCAUUCGGA-3’ Direction of Transcription

Drawing Exercise In reading literature, you run into a research article depicting a gene structure as follow (boxes represent exons). Label the location of promoter, transcriptional start site (TSS), and introns on the figure. Draw a picture of the mRNA product and the mature mRNA after processing. Exon 1Exon 2Exon 3 Exon 4

Drawing Exercise In reading literature, you run into a research article depicting a gene structure as follow (boxes represent exons). Label the location of promoter, transcriptional start site (TSS), and introns on the figure. Draw a picture of the mRNA product and the mature mRNA after processing. Exon 1Exon 2Exon 3 Exon 4 Promoter Exon 1Exon 2Exon 3 Exon 4 Exon 1Exon 2Exon 3Exon 4 Red lines indicate introns Transcription RNA Processing CAP Poly A TSS Exon 1Exon 2Exon 3 Exon 4 The first nucleotide of exon 1 is the transcriptional start site (TSS) and DNA upstream from TSS is the promoter. After transcription, the RNA is produced and regions in between the exons are introns (indicated by red). Note that in this exercise, double stranded DNA and single stranded RNA are not distinguished by double lines versus single line. Answer:

Section IV: A few Concepts Modularity: Transcription factors are composed of functional domains, usually at least a DNA binding domain and a transcriptional regulatory domain (either an activation domain or repressor domain). These domains can be moved from one protein to another protein and are still functional (that is, modular domain). The combinatorial mechanism: Promoter is made of various binding sites. These binding sites are not unique to any given promoter. Instead, they are found in a variety of promoters. As an example, a liver specific-gene and a pancreas-specific gene may share some common binding sites in their promoters. This raises an important question: How is tissue-specific transcription achieved? The current model is that this is achieved through a "combinatorial mechanism”: The figure below depicts two promoters sharing some common cis-acting elements (shaded red), but these elements are combined with different binding sites (unshaded boxes or circles) on each promoter. Binding of the corresponding transcription factors to these sites determines the activity of the given promoter. The reason that these two promoters are turned on in different tissues/cell types is that different cells have different “sets of transcription factors.” One way to think about this is that the transcription factors in a given cell type constitute “a combination of passwords” that cooperatively open the lock. Another way to think about this is that each promoter is like a jigsaw puzzle. Only when a given cell contains the correct pieces (the correct transcription factors) can it solve the puzzle (switch on the gene). A. B.

Families of transcription factors: By convention, the sequence-specific transcription factors have been classified into “families” on the basis of their DNA binding domains, which allow them to recognize and bind to a “consensus sequence.” Click the “Attachments” (on the upper right corner) for some examples of sequence-specific transcription factors. Genetic flow of information—the central dogma: The flow of sequence information from genomic DNA to mRNA to protein is the “central dogma” of molecular biology. transcription translation DNA  RNA  Protein Section IV: A few Concepts (Cont’d)

Reverse transcription: The discovery of retrovirus and its ability to make DNA using RNA as template indicated that the central dogma is not always correct. This revolutionized the field. The enzyme that makes DNA using RNA as template is called reverse transcriptase and the process called reverse transcription. Some retroviruses cause cancer, because they carry the cancer causing genes called “oncogenes.” Examples of retrovirus include human T-lymphotropic virus (HTLV, causes T cell lymphoma and Tropical Spastic Paraparesis) and human immunodeficiency virus (HIV, causes AIDS). RNA replication in the figure on the left below shows that RNA can also be made using RNA as template. RNA genome Chromosome Nucleus Double stranded viral DNA RNA entering into the host cells Host Cells Reverse Transcription Viral DNA integrated into the chromosome Retrovirus

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