Prokaryotic Transcription

Transcription in Prokaryotes

The genetic code is a degenerate, non-overlapping set of 64 codons that encodes for 21 amino acids and 3 stop codons.

Learning Objectives

Describe the genetic code and how the nucleotide sequence prescribes the amino acid and the protein sequence

Key Takeaways

Key Points

  • The relationship between DNA base sequences and the amino acid sequence in proteins is called the genetic code.
  • There are 61 codons that encode amino acids and 3 codons that code for chain termination for a total of 64 codons.
  • Unlike, eukayrotes, a bacterial chromosome is a covalently-closed circle.
  • The DNA double helix must partially unwind for transcription to occur; this unwound region is called a transcription bubble.

Key Terms

  • nucleotide: the monomer comprising DNA or RNA molecules; consists of a nitrogenous heterocyclic base that can be a purine or pyrimidine, a five-carbon pentose sugar, and a phosphate group
  • amino acid: Any of 20 naturally occurring α-amino acids (having the amino, and carboxylic acid groups on the same carbon atom), and a variety of side chains, that combine, via peptide bonds, to form proteins.
  • redundancy: duplication of components, such as amino acid codons, to provide survival of the total system in case of failure of single components

The Genetic Code: Nucleotide sequences prescribe the amino acids

The genetic code is the relationship between DNA base sequences and the amino acid sequence in proteins. Features of the genetic code include:

  • Amino acids are encoded by three nucleotides.
  • It is non-overlapping.
  • It is degenerate.

There are 21 genetically-encoded amino acids universally found in the species from all three domains of life. ( There is a 22nd genetically-encooded amino acid, Pyl, but so far it has only been found in a handful of Archaea and Bacteria species.) Yet there are only four different nucleotides in DNA or RNA, so a minimum of three nucleotides are needed to code each of the 21 (or 22) amino acids. The set of three nucleotides that codes for a single amino acid is known as a codon. There are 64 codons in total, 61 that encode amino acids and 3 that code for chain termination. Two of the codons for chain termination can, under certain circumstances, instead code for amino acids.

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Genetic Code Table.: A codon is made of three nucleotides. Consequently there are 43 (=64) different codons. The 64 codons encode 22 different amino acids and three termination codons, also called stop codons.

Degeneracy is the redundancy of the genetic code. The genetic code has redundancy, but no ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them specifies any other amino acid (no ambiguity). The codons encoding one amino acid may differ in any of their three positions. For example, the amino acid glutamic acid is specified by GAA and GAG codons (difference in the third position); the amino acid leucine is specified by UUA, UUG, CUU, CUC, CUA, CUG codons (difference in the first or third position); while the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (difference in the first, second or third position). These properties of the genetic code make it more fault-tolerant for point mutations.

Origin of transcription on prokaryotic organisms

Prokaryotes are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. The central region of the cell in which prokaryotic DNA resides is called the nucleoid region. Bacterial and Archaeal chromosomes are covalently-closed circles that are not as extensively compacted as eukaryotic chromosomes, but are compacted nonetheless as the diameter of a typical prokaryotic chromosome is larger than the diameter of a typical prokaryotic cell. Additionally, prokaryotes often have abundant plasmids, which are shorter, circular DNA molecules that may only contain one or a few genes and often carry traits such as antibiotic resistance.

Transcription in prokaryotes (as in eukaryotes) requires the DNA double helix to partially unwind in the region of RNA synthesis. The region of unwinding is called a transcription bubble. Transcription always proceeds from the same DNA strand for each gene, which is called the template strand. The RNA product is complementary to the template strand and is almost identical to the other (non-template) DNA strand, called the sense or coding strand. The only difference is that in RNA all of the T nucleotides are replaced with U nucleotides.

The nucleotide on the DNA template strand that corresponds to the site from which the first 5′ RNA nucleotide is transcribed is called the +1 nucleotide, or the initiation site. Nucleotides preceding, or 5′ to, the template strand initiation site are given negative numbers and are designated upstream. Conversely, nucleotides following, or 3′ to, the template strand initiation site are denoted with “+” numbering and are called downstream nucleotides.

Initiation of Transcription in Prokaryotes

RNA polymerase initiates transcription at specific DNA sequences called promoters.

Learning Objectives

Summarize the initial steps of transcription in prokaryotes

Key Takeaways

Key Points

  • Transcription of mRNA begins at the initiation site.
  • Two promoter consensus sequences are at the -10 and -35 regions upstream of the initiation site.
  • The σ subunit of RNA polymerase recognizes and binds the -35 region.
  • Five subunits (α, α, β, β’, and σ) make up the complete RNA polymerase holoenzyme.

Key Terms

  • holoenzyme: a fully functioning enzyme, composed of all its subunits
  • promoter: the section of DNA that controls the initiation of RNA transcription

Prokaryotic RNA Polymerase

Prokaryotes use the same RNA polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five polypeptide subunits, two of which are identical. Four of these subunits, denoted α, α, β, and β’, comprise the polymerase core enzyme. These subunits assemble each time a gene is transcribed; they disassemble once transcription is complete. Each subunit has a unique role: the two α-subunits are necessary to assemble the polymerase on the DNA; the β-subunit binds to the ribonucleoside triphosphate that will become part of the nascent “recently-born” mRNA molecule; and the β’ binds the DNA template strand. The fifth subunit, σ, is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites and would produce mRNA molecules that specified protein gibberish. The polymerase comprised of all five subunits is called the holoenzyme.

Prokaryotic Promoters and Initiation of Transcription

The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5′ mRNA nucleotide is transcribed is called the +1 site, or the initiation site. Nucleotides preceding the initiation site are given negative numbers and are designated upstream. Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called downstream nucleotides.

A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In most cases, promoters exist upstream of the genes they regulate. The specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, some of the time, or infrequently. Although promoters vary among prokaryotic genomes, a few elements are conserved. At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized and bound by σ. Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region facilitates unwinding of the DNA template; several phosphodiester bonds are made. The transcription initiation phase ends with the production of abortive transcripts, which are polymers of approximately 10 nucleotides that are made and released.

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Promoter: The σ subunit of prokaryotic RNA polymerase recognizes consensus sequences found in the promoter region upstream of the transcription start sight. The σ subunit dissociates from the polymerase after transcription has been initiated.

Elongation and Termination in Prokaryotes

Transcription elongation begins with the release of the polymerase σ subunit and terminates via the rho protein or via a stable hairpin.

Learning Objectives

Explain the process of elongation and termination in prokaryotes

Key Takeaways

Key Points

  • The transcription elongation phase begins with the dissociation of the σ subunit, which allows the core RNA polymerase enzyme to proceed along the DNA template.
  • Rho-dependent termination is caused by the rho protein colliding with the stalled polymerase at a stretch of G nucleotides on the DNA template near the end of the gene.
  • Rho-independent termination is caused the polymerase stalling at a stable hairpin formed by a region of complementary C–G nucleotides at the end of the mRNA.

Key Terms

  • elongation: the addition of nucleotides to the 3′-end of a growing RNA chain during transcription

Elongation in Prokaryotes

The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core RNA polymerase enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. Since the base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components, RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

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Elongation in prokaryotes: During elongation, the prokaryotic RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5′ to 3′ direction, and unwinds and rewinds the DNA as it is read.

Termination in Prokaryotes

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly-made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals: one is protein-based and the other is RNA-based.

Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.

Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides. The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.

Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently in the cytoplasm. The unification of transcription, translation, and even mRNA degradation is possible because all of these processes occur in the same 5′ to 3′ direction and because there is no membranous compartmentalization in the prokaryotic cell. In contrast, the presence of a nucleus in eukaryotic cells prevents simultaneous transcription and translation.