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15.2B: Initiation of Transcription in Prokaryotes - Biology

15.2B: Initiation of Transcription in Prokaryotes - Biology


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RNA polymerase initiates transcription at specific DNA sequences called promoters.

Learning Objectives

  • Summarize the initial steps of transcription in prokaryotes

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.


15.2B: Initiation of Transcription in Prokaryotes - Biology

The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. A bacterial chromosome is a covalently closed circle that, unlike eukaryotic chromosomes, is not organized around histone proteins. The central region of the cell in which prokaryotic DNA resides is called the nucleoid. In addition, prokaryotes often have abundant plasmids, which are shorter circular DNA molecules that may only contain one or a few genes. Plasmids can be transferred independently of the bacterial chromosome during cell division and often carry traits such as antibiotic resistance. Because of these unique features, transcription and gene regulation is somewhat different between prokaryotic cells and eukaryotic ones.

Learning Objectives

  • Understand the basic steps in the transcription of DNA into RNA in prokaryotic cells
  • Understand the basics of prokaryotic translation and how it differs from eukaryotic translation

The Central Dogma: DNA Encodes RNA RNA Encodes Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma (Figure 9.14), which states that genes specify the sequences of mRNAs, which in turn specify the sequences of proteins.

Figure 9.14 The central dogma states that DNA encodes RNA, which in turn encodes protein.

The copying of DNA to mRNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every complementary nucleotide read in the DNA strand. The translation to protein is more complex because groups of three mRNA nucleotides correspond to one amino acid of the protein sequence. However, as we shall see in the next module, the translation to protein is still systematic, such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.


Termination

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by protein release factors that resemble tRNAs. The releasing factors in both prokaryotes and eukaryotes instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 3 and listed in Table 1.

Figure 3. (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these processes and necessitating RNA processing for stability.


Elongation and Termination in Prokaryotes

The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core 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 (Figure 2). The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

Figure 2. Click for a larger image. 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.


The Structures of Eukaryotic Transcription Pre-initiation Complexes and Their Functional Implications

Transcription is a highly regulated process that supplies living cells with coding and non-coding RNA molecules. Failure to properly regulate transcription is associated with human pathologies, including cancers. RNA polymerase II is the enzyme complex that synthesizes messenger RNAs that are then translated into proteins. In spite of its complexity, RNA polymerase requires a plethora of general transcription factors to be recruited to the transcription start site as part of a large transcription pre-initiation complex, and to help it gain access to the transcribed strand of the DNA. This chapter reviews the structure and function of these eukaryotic transcription pre-initiation complexes, with a particular emphasis on two of its constituents, the multisubunit complexes TFIID and TFIIH. We also compare the overall architecture of the RNA polymerase II pre-initiation complex with those of RNA polymerases I and III, involved in transcription of ribosomal RNA and non-coding RNAs such as tRNAs and snRNAs, and discuss the general, conserved features that are applicable to all eukaryotic RNA polymerase systems.

Keywords: Cryo-electron microscopy Gene expression General transcription factors Initiation RNA polymerase Structural biology TFIID TFIIH Transcription.

Figures

Architecture of multisubunit RNA polymerases…

Architecture of multisubunit RNA polymerases across kingdoms and organization of eukaryotic Pol II…

Architecture of eukaryotic RNA polymerase…

Architecture of eukaryotic RNA polymerase II. a, b Depiction of transcribing Pol II…

The structure of human TFIID.…

The structure of human TFIID. a Organization of human TFIID into three lobes.…

Proposed conformational transitions of TFIID…

Proposed conformational transitions of TFIID during DNA binding and mechanism of TBP deposition.…

Structure and function of TFIIH.…

Structure and function of TFIIH. a Schematic of TFIIH architecture. Main enzymatic subunits…

The structure of the Pol II-PIC and its components. a Structure of promoter…

Structure of the open and…

Structure of the open and initially transcribing human Pol II-PICs and comparison with…

The Mediator-bound Pol II-PIC. a…

The Mediator-bound Pol II-PIC. a Crystal structure of the Mediator head and middle…

Architectural model of the holo-Pol…

Architectural model of the holo-Pol II-PIC with TFIID and Mediator. Proposed model of…

The architecture of the RNA…

The architecture of the RNA polymerase I PIC and ITC. a Structure of…

Comparison of DNA paths in…

Comparison of DNA paths in the Pol I and Pol II ITCs. a…

Transcription factor-like subunits of Pol…

Transcription factor-like subunits of Pol III. a Structure of Pol I extracted from…

The structure of TFIIIB. a…

The structure of TFIIIB. a Superposition of the structures of the DNA-bound TBP-Brf2…

The structure of the Pol III-ITC. a Overview of the structure of the…

Comparison of DNA-bound structures of…

Comparison of DNA-bound structures of TFIIB-like initiation factors. a–c Structure of the Pol…


Watch the video: Osmosis Jones meets Memento, cell needs help remebering prokaryotic transcription (May 2022).