3 Transcription

Learning Objectives

Upon completing this chapter, you will be able to:

• Define transcription and describe the basic structure and composition of an RNA transcript.
• Distinguish between the DNA template (antisense) strand and the nontemplate (sense) strand.
• Describe the key components and function of bacterial RNA polymerase, including the role of the sigma (σ) factor.
• Outline the three main stages of transcription in bacteria: initiation, elongation, and termination.
• Explain the role of promoters and terminators in gene transcription.
• Compare and contrast RNA polymerase with DNA polymerase.

Introduction to Transcription

The genetic information stored in DNA directs the synthesis of proteins, but DNA does not directly participate in protein synthesis. Instead, an intermediary molecule, ribonucleic acid (RNA), is created. During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an RNA transcript. This resulting single-stranded RNA molecule is composed of ribonucleotides containing the bases adenine (A), cytosine (C), guanine (G), and uracil (U). It acts as a mobile molecular copy of the original DNA sequence for a specific gene.

For transcription to occur, the DNA double helix must partially unwind in the region of RNA synthesis, forming a temporary transcription bubble. Transcription of a particular gene always proceeds from one of the two DNA strands, which acts as the template strand (also known as the antisense strand). The RNA product is complementary to this DNA template strand. Consequently, the RNA sequence is almost identical to the other DNA strand, the nontemplate DNA strand (or sense strand). The key difference is that in RNA, all thymine (T) nucleotides found in the DNA sense strand are replaced with uracil (U) nucleotides. During RNA synthesis, U is incorporated when there is an A in the complementary DNA template (antisense) strand.

The Transcription Machinery in Bacteria

Bacteria utilize a single type of RNA polymerase to transcribe all of their genes. Like DNA polymerase, RNA polymerase adds nucleotides one by one to the 3’-OH group of the growing nucleotide chain, forming a phosphodiester bond by dehydration synthesis between the new nucleotide and the last one added.

However, there are critical differences:

• Primer Requirement: DNA polymerase requires a free 3’-OH group (provided by a primer) to begin synthesis. In contrast, RNA polymerase does not require a primer and can initiate RNA synthesis de novo.
• Substrate: DNA polymerase uses deoxyribonucleotides, while RNA polymerase uses ribonucleotides.

In E. coli, the RNA polymerase holoenzyme (the complete, active enzyme) comprises six polypeptide subunits. Five of these subunits (α2​ββ′ω) make up the core enzyme, which is responsible for the catalytic activity of adding RNA nucleotides to a growing strand. The sixth subunit is known as the sigma (σ) factor. The σ factor is crucial for the initiation of transcription as it enables the RNA polymerase holoenzyme to recognize and bind specifically to promoter sequences on the DNA, thus allowing for the transcription of particular genes. Bacteria possess various σ factors, each enabling the polymerase to recognize different sets of promoters, thereby allowing for the differential transcription of various genes under different conditions.

Initiation: Starting Transcription

The initiation of transcription begins when the RNA polymerase holoenzyme binds to a specific DNA sequence called a promoter. The promoter is typically located just upstream (before) the gene it regulates.

• Promoter Elements: The DNA sequence of a promoter contains specific recognition sites for the RNA polymerase. The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5’ RNA nucleotide is transcribed is called the initiation site, designated as +1. Nucleotides preceding the initiation site are designated with negative numbers (“upstream”), whereas nucleotides following the initiation site are called “downstream” nucleotides and are designated with positive numbers.
• Consensus Sequences: While promoter sequences can vary among bacterial genes, two short DNA sequences within the promoter region are highly conserved across various bacterial species. These are known as consensus sequences:
  • The -10 consensus sequence (often called the Pribnow box or TATA box in bacteria) is typically TATAAT and is located about 10 base pairs upstream from the +1 initiation site.
  • The -35 consensus sequence (typically TTGACA) is located approximately 35 base pairs upstream from the +1 site. The σ factor of the RNA polymerase primarily recognizes and binds to this -35 sequence.

Once the RNA polymerase holoenzyme, guided by the σ factor, binds to the promoter, it unwinds the DNA at the -10 region (transcription bubble formation) and is positioned to begin transcribing the gene from the +1 site.

Elongation: Synthesizing the RNA Transcript

The elongation in transcription phase begins once the RNA polymerase has synthesized a short stretch of RNA (about 8-10 nucleotides). At this point, the σ subunit dissociates from the RNA polymerase. The remaining core enzyme then moves along the DNA template, synthesizing RNA complementary to the template strand in the 5’ to 3’ direction.

• Processivity: As the core enzyme proceeds, it continuously unwinds the DNA double helix ahead of it and rewinds the DNA behind it after the RNA has been synthesized (Figure 3.1).
• Rate: In bacteria like E. coli, RNA synthesis during elongation occurs at a rate of approximately 40 nucleotides per second.

Termination: Ending Transcription

Once a gene is fully transcribed, the bacterial RNA polymerase must dissociate from the DNA template and liberate the newly made RNA transcript. This process is referred to as termination of transcription.

• Termination Signals: The DNA template includes specific repeated nucleotide sequences called the terminator that act as termination signals. These signals, when transcribed into RNA, cause the RNA polymerase to stall.
• Release: Following stalling, the RNA polymerase releases from the DNA template, and the newly synthesized RNA transcript is freed. There are two main mechanisms of termination in bacteria: Rho-dependent and Rho-independent termination (though the provided text focuses on the general concept of termination signals leading to release).

The released RNA transcript can then, if it’s a messenger RNA (mRNA), proceed to the next stage of gene expression: translation.

Chapter Summary

Transcription is the cellular process of synthesizing an RNA transcript from a DNA template. In bacteria, this is carried out by the enzyme RNA polymerase. The process begins with initiation, where the RNA polymerase holoenzyme, containing a σ factor, recognizes and binds to specific promoter sequences (like the -10 and -35 regions) on the DNA, upstream of a gene. Unlike DNA polymerase, RNA polymerase does not require a primer. During elongation, the σ factor detaches, and the RNA polymerase core enzyme moves along the DNA template, unwinding it and synthesizing a complementary RNA strand in the 5’ to 3’ direction, using uracil (U) in place of thymine (T). Finally, termination occurs when the RNA polymerase encounters the terminator or termination signals in the DNA, causing it to dissociate and release the completed RNA transcript. This RNA molecule then serves as a mobile copy of the genetic information.