General Genetics/Transcription

Transcription is the process by which the genetic information in DNA is converted into RNA. The mechanism of transcription can be described simply in 6 steps (5 in prokaryotes):

1. The section of DNA to be transcribed is recognized by proteins involved in transcription.
2. Hydrogen bonds between base pairs of DNA are broken and the DNA double helix "unzips."
3. Complementary RNA bases bind to the now exposed DNA bases
4. RNA Polymerase binds the RNA bases together to form a strand of mRNA
5. Hydrogen bonds formed between the original DNA strand and the new RNA strand are broken
6. in eukaryotes, the new RNA strand is further processed and moves to the cytoplasm.

The segment of DNA to be transcribed is called a transcription unit, and contains at least one gene. If this gene codes for a protein, the RNA transcription product is called Messenger RNA, or mRNA. The gene may also code for Ribosomal RNA (rRNA), transfer RNA (tRNA), or a ribozyme.

Prokaryotic Transcription

Anatomy of the Typical Prokaryotic Gene

The promoter lies upstream of the gene. The end of the promoter is the transcription initiation site, where the DNA sequence to be transcribed begins. The terminator sequence stops transcription. Only two regions tend to be conserved in prokaryotic promoter, a site called the Pribnow box 10 bp upstream of the gene and a site 35 bp upstream of the gene. The -35 site (consensus sequence: TTGAC) is where the RNA polymerase binds, and it first melts the strands at the Pribnow box (consensus sequence: TATAAT).

There is not a specific conserved or consensus sequence that characterizes a prokaryotic terminator sequence. However, they are characteristically palindromic: from a central point of symmetry, the sequence is complementary to itself. Transcription of the sequence forms an RNA transcript that folds into a "hairpin" secondary structure. The hairpin "clogs" the polymerase.

A Rho-dependent termination requires an enzyme Rho which follows the transcription. When RNA polymerase pauses due to the hairpin structure, Rho uses its helicase-like functionality to remove the RNA transcript from DNA. In the absence of Rho, terminator sequences are often followed by a series of A nucleotides. Because the intermolecular forces between A and U are relatively weak, less energy is required to remove the transcript from the DNA strand.

Prokaryotic RNA Polymerase

Prokaryotic RNA polymerase covers about 60 bp. It has helicase-like activity, meaning that it is capable of unwinding the DNA strands. Ribo-NTPs enter the RNA polymerase and hydrogen-bond to the nucleotides of the coding DNA strand. The polymerase catalyzes the formation of a hydrogen bond between the 3' hydroxyl group of the last

E. coli RNA polymerase consists of a core with four subunits: β, β', and two α subunits. Addition of a σ protein, which is responsible for binding-site recognition, creates a functional holoenzyme.

Due to the lack of a nucleus and lack of RNA processing in prokaryotes, prokaryotes are capable of "coupled" transcription, where ribosomes begin creating proteins from the mRNA before DNA polymerase is finished synthesizing the mRNA from the DNA template.

Eukaryotic Transcription

Eukaryotic RNA Polymerases

RNA Pol I is located in the nucleolus and transcribes certain rRNA.

RNA Pol II is located in the nucleus and transcribes mRNA and most SnRNA.

RNA Pol III is located in the nucleus and transcribes tRNA, certain rRNA, and some SnRNA.

Eukaryotic Gene Expression

The typical eukaryotic Pol II promoter has the CAAT box (sequence: GGNCAATCT) located 75 bp upstream of the gene and the TATA or Hogness box (sequence: TATAAA) located 25 bp upstream. However, RNA Pol II cannot bind directly to the promoter. There is an entire complex of transcription factors that are required to bind to Pol II before the polymerase can even bind to the DNA. This transcription factor complex is known as the preinitiation complex. Each cell type has its own characteristic transcription factors that lead to distinctly different expressions of the same genome.

Eukaryotes also have tissue-specific enhancers (which upregulate expression of a gene) and silencers (which downregulate expression of a gene). Homodimeric activator proteins have high affinity for enhancer sequences. The activator recruits a complex of proteins, which exert two functions: (1) RNA Pol II binds is bound to the enhancer site and (2) the DNA folds such that the RNA polymerase "lands" on the gene that the enhancer upregulates. The polymerase then transcribes the gene. Enhancers can be hundreds of thousands of base pairs away from the gene whose expression they upregulate.

Termination of Eukaryotic Transcription: The "Torpedo Model"

At the polyadenylation cleavage site, the cleavage and polyadenylation (CPA) complex is recruited and binds to RNA Pol II. This allows cleavage and slows RNA Pol II. RNA transcribed after cleavage invades the DNA duplex and forms an "R loop" which further impedes the velocity of RNA Pol II. The Xrn2 exonuclease attaches at the 5' end and degrades the transcript. SETX helicase acts as a "torpedo" to catch the paused RNA Pol II, and all components release.