Why this lecture matters

DNA is valuable because it stores genetic information, but information is useful only if a cell can access it at the right time and in the right place. In the previous lecture, we focused on how DNA can be copied and protected. In this lecture, we shift from copying DNA for inheritance to using DNA information for cell function. The first major step in gene expression is transcription: the process of making an RNA copy from a DNA template.

Transcription is central to biology because it connects stored information to cellular activity. A gene is a stretch of DNA that encodes a functional product. Many genes encode proteins, but some genes encode functional RNA molecules. In either case, the cell must decide which gene to express, where transcription should begin, which DNA strand should be used as the template, and where transcription should end. These choices are not contained only in the protein-coding sequence. They depend on additional DNA sequences, such as promoters and terminators, and on proteins that recognize these sequences.

The central dogma: information flow in cells

The central dogma describes the usual flow of genetic information: DNA is transcribed into RNA, and RNA can be translated into protein (Figure 1). Protein function then contributes to cellular function and phenotype. DNA is the long-term storage form of genetic information. RNA is often a working copy of selected information. Proteins carry out many cellular functions, including catalysis, structure, transport, signaling, and regulation.

This model is a powerful simplification, but it is not the whole story. One important exception is reverse transcription, in which RNA is copied back into DNA. Telomerase, discussed in the previous lecture, is one example of an enzyme with reverse transcriptase activity. In addition, not every RNA is translated into protein. Transfer RNA, ribosomal RNA, microRNAs, and spliceosome RNAs are examples of RNAs that function as RNA molecules. Therefore, a more complete way to think about the central dogma is that DNA information can be copied into RNA, and RNA can either function directly or be used to make protein.

Key idea: Transcription makes RNA copies of selected DNA information. These RNA molecules can be translated into proteins or function directly as RNA.

Figure 1. The central dogma describes the flow of genetic information. Genes are stretches of DNA arranged on chromosomes. During transcription, RNA is made as a copy of a selected stretch of DNA sequence. For protein-coding genes, the RNA sequence is then used to make a linear protein sequence. The protein’s amino acid sequence determines its function, and protein function contributes to the phenotype of the organism. An important exception to the usual DNA → RNA flow is reverse transcription, in which RNA is copied back into DNA, as occurs with reverse transcriptase enzymes such as telomerase. Attribution: Satoshi H. Namekawa

The Challenge of Transcription: Making the Right RNA Copy

At first, transcription might sound simple: use DNA as a template and make RNA. But a cell faces several specific problems. First, the genome is very large, so the cell must identify the correct gene to transcribe. Second, DNA is double-stranded, but only one strand is used as the template for a given gene. Third, RNA polymerase must know which direction to move. Fourth, transcription must start and stop at appropriate places. Finally, the cell must regulate how much RNA is made, because gene expression affects cell behavior.

Promoters are DNA sequences that help solve the starting problem. A promoter is a site where transcription machinery binds. The transcription start site is the position where RNA synthesis begins, often labeled +1. Terminators, or termination signals, help solve the stopping problem. These regulatory sequences are part of the information stored in DNA, even though they do not necessarily encode amino acids. In this sense, DNA stores both product information and control information.

RNA building blocks and the energy story

RNA and DNA are chemically similar polymers, but they differ in important ways (Figure 2). RNA uses ribose sugar, whereas DNA uses deoxyribose. RNA uses uracil instead of thymine. RNA is also usually single-stranded, while DNA is usually double-stranded. These differences help explain why RNA can serve as a flexible, temporary working copy of genetic information.

The building blocks for RNA synthesis are ribonucleoside triphosphates: ATP, UTP, GTP, and CTP. RNA polymerase adds these ribonucleotides to the growing RNA chain. Like DNA synthesis, RNA synthesis proceeds in the 5' to 3' direction. Each new nucleotide is added to the 3' end of the growing RNA. The energy for building the polymer comes from the triphosphate groups on the incoming ribonucleotides. When a nucleotide is added, high-energy phosphate bonds are broken, helping drive polymer formation. Unlike DNA polymerase, RNA polymerase does not require a primer to begin RNA synthesis.

Figure 2. Basic chemical components of nucleotides. DNA and RNA are polymers made from nucleotide building blocks. Each nucleotide contains three parts: a nitrogenous base, a sugar, and one or more phosphate groups. The nitrogenous bases include purines, adenine and guanine, and pyrimidines, cytosine, thymine, and uracil. DNA uses deoxyribose sugar and the bases A, T, G, and C, whereas RNA uses ribose sugar and the bases A, U, G, and C. The phosphate groups link nucleotides together to form the sugar-phosphate backbone of a nucleic acid polymer. Attribution: Marc T. Facciotti 

Initiation: starting transcription

Transcription initiation is the step in which RNA polymerase is positioned at the correct gene and begins RNA synthesis. In bacteria, a sigma factor helps RNA polymerase recognize promoter DNA (Figure 4, Top). Many bacterial promoters contain conserved sequences near -35 and -10 relative to the transcription start site. A common bacterial -10 element is similar to TATAAT. These promoter sequences help position RNA polymerase and influence how frequently a gene is transcribed.

In eukaryotes, transcription initiation is more complex. Eukaryotic cells have several RNA polymerases. In this lecture, we focus on RNA polymerase II because it transcribes most protein-coding genes, producing pre-mRNAs that are later processed into mature mRNAs. Other RNA polymerases make other types of RNA, such as rRNA and tRNA. RNA polymerase II does not simply bind promoters by itself. General transcription factors help assemble a pre-initiation complex at the promoter and recruit RNA polymerase II (Figure 4, Bottom). Some eukaryotic promoters contain a TATA box, but only a minority of human genes have a TATA-box promoter. The broader point is that promoter recognition is required in both bacteria and eukaryotes, but the molecular details differ.

Once the transcription machinery is assembled, the DNA near the start site unwinds. One DNA strand becomes the template strand. RNA polymerase reads this template strand in the 3' to 5' direction and synthesizes a complementary RNA molecule in the 5' to 3' direction. After initiation, RNA polymerase leaves the promoter and enters elongation.

After initiation, transcription enters elongation. Although bacteria and eukaryotes use different proteins and regulatory details, the basic logic of elongation is shared. In both groups, RNA polymerase moves along one DNA template strand, locally unwinds DNA, and synthesizes RNA in the 5′ to 3′ direction. The open region of DNA is called a transcription bubble. Within this bubble, RNA nucleotides base-pair with the DNA template strand. As RNA polymerase moves forward, DNA ahead of the enzyme unwinds and DNA behind the enzyme rewinds. The RNA transcript peels away from the DNA as it is synthesized.

The RNA product is complementary to the DNA template strand. It is also similar to the coding strand, except that RNA contains uracil instead of thymine. This relationship is important when predicting RNA sequences from DNA sequences. If you are given the coding strand, the RNA sequence will match it except U replaces T. If you are given the template strand, the RNA sequence must be complementary and antiparallel.

Termination also follows a shared principle in bacteria and eukaryotes: transcription must stop at the correct region rather than continuing randomly forever. The exact termination mechanisms differ between organisms and genes, but in all cases, specific DNA or RNA signals help end transcription. During termination, RNA polymerase stops, the newly synthesized RNA is released, and RNA polymerase separates from the DNA template.