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Lecture 8

Transcription (RNA Synthesis)

The Central Dogma states that DNA is used to make RNA, which in turn is used to make proteins. We know now, of course, that DNA can also be made from RNA with reverse transcriptase.

Transcription can be defined as the process of making RNA from DNA. Enzymes responsible for making RNA from DNA are called RNA polymerases. These enzymes use ribonucleoside triphosphates and DNA to create RNA molecules (called transcripts). The portion of DNA copied is referred to as the template. Like DNA synthesis, transcription proceeds in the 5' to 3' direction. RNA polymerases differ from DNA polymerases in a couple of important respects. First, RNA polymerases do not require a primer. Second, RNA polymerases are probably much less accurate in their copying than are DNA polymerases because they do not have proofreading ability (3 ' to 5 ' exonuclease)

There are three major kinds of RNA in all cells: ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). All of the major RNAs have important roles in the process of converting nucleotide sequence information into proteins (protein synthesis or translation).

 RNA molecules in E. coli
Type Abbreviation
Function

Size in nucleotides

Transfer RNA tRNA Carries activated amino acid

about 75

Ribosomal RNA 5S rRNA
16S rRNA
23S rRNA
Ribosome component
Ribosome component
Ribosome component

 120
1542
2904

Messenger RNA mRNA Codes for proteins

variable

Bacterial (prokaryotic) cells have a single RNA polymerase that makes all three types of RNA in the cell. Higher cells (eukaryotic cells) have three types of RNA polymerase (called RNA polymerase I, RNA polymerase II, and RNA polymerase III). In eukaryotes, each type of RNA is made by its own polymerase:

RNA polymerase I makes rRNA
RNA polymerase II makes mRNA
RNA polymerase III makes tRNA

E. coli RNA Polymerase

E. coli RNA polymerase has the following subunit composition: two alpha, one beta, one beta prime, and one sigma subunit constitute the RNA polymerase holoenzyme. RNA polymerase lacking sigma subunit is called the core RNA polymerase. RNA is synthesized in the 5' to 3' direction (the same direction as DNA is synthesized). The synthesis of RNA does not require a primer, but does require a DNA template strand. The first nucleotide of the RNA chain retains the 5'-triphosphate group, but all subsequent nucleotides that are added to the growing chain only retain the alpha phosphate in the phosphodiester linkage.

The overall reaction is: n rNTP ----> (rNMP)n + n PPi

Transcription can be divided into three phases: initiation, elongation, and termination

Initiation


Initiation of transcription occurs at special sites on DNA called promoters (
FIGURE). In bacteria, promoters are typically short sequences located at the -35 region (TTGACA) and the -10 region (TATAAT, often called a TATA box) to which RNA polymerase binds. Promoters are close (within 10 base pairs) to the location of the first nucleotide, which is called the "start site" of transcription. In E. coli, a factor called sigma binds directly to RNA polymerase and helps the polymerase to bind to promoter sequences. The binding of RNA polymerase at the promoter opens the DNA at the AT-rich TATAAT sequence so that a transcription bubble about 17 bases long forms. Some genes are always "on" (constitutively expressed). Some genes may be constitutively expressed at low levels (weak promoters) or at high levels (strong promoters). RNA polymerase may use other proteins (transcription factors or activators) to help the enzyme to recognize and bind to promoters. Other proteins may bind near the promoter to block the binding of RNA polymerase; such proteins are called "repressors". Activators and repressors regulate (or control) the process of transcription. Therefore, under certain circumstances, genes can be "turned on" or "turned off". Turning on a gene means to activate transcription of the gene or "express" the gene. Turning off a gene means to inhibit transcription of a gene. It is important to realize that a cell has many genes, but not all of them are turned on or off at the same time. For many genes in the cell, it is critical that the proper amount of a particular protein is made and that it is made at the right time. The consequences of either having a protein missing when it is needed, or of having too much of it can be severe - cell death or tumor formation in some cases. Mutation is therefore important not just because it can affect the DNA sequence (and thus amino acid sequence) of an important enzyme, but also because a mutation can affect a control sequence, such as a promoter and alter when a gene is turned on or how much RNA is synthesized.

Elongation (FIGURE)

Once transcription has started, sigma factor is released by RNA polymerase. As transcription proceeds, DNA ahead of the transcription bubble is overwound, and DNA behind the transcription bubble is underwound. Gyrase (topoisomerase II) relieves overwinding and topoisomerase I relieves underwinding. Only a portion of the newly synthesized (nascent) RNA molecule remains base-paired to DNA as it is being made. The 5' end becomes free, and is available for action by other proteins. In bacteria, for example, translation can begin on the 5' free end of a mRNA as it is being made. Thus translation and transcription in bacteria can be coupled together. Such is not the case in eukaryotes because transcription occurs in the nucleus and translation occurs in the cytoplasm.

Termination

Just as RNAs have specific start points defined by promoters, sequence information within the DNA also specifically signals the stop of transcription. Termination is important not only for defining the length of RNAs made, but also for release (removal of base pairing) of the RNA from the DNA. Less is known about termination of transcription than initiation. In bacteria, two termination mechanisms are well known. They are called intrinsic termination and rho-dependent termination. Intrinsic termination involves sequences within the RNA as it is being made that signal the RNA polymerase to stop. Such sequences form a stem-loop (where the stem is GC-rich) and a string of U's (templated by a string of A's). (see FIGURE). Rho-dependent termination uses a protein called rho-factor to stop RNA sysnthesis at specific sites.