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Bacterial mRNA usually has a shorter lifespan

Posted by star on 2018-10-25 19:15:49

    An important characteristic of bacterial mRNA is that its lifetime is short compared to other types of bacterial RNA molecules. The half-life of a typical bacterial mRNA molecule is a few minutes. This feature, which may seem terribly wasteful, has an important regulatory function. A cell can turn off the synthesis of a protein that is no longer needed by turning off synthesis of the mRNA that encodes the protein. Soon after, none of that particular mRNA will remain and synthesis of the protein will cease. Of course, this regulation also means that in order to maintain synthesis of a particular protein, the mRNA molecules encoding these proteins must be synthesized continuously. Continuous mRNA synthesis is a small payment by the cell for the ability to regulate the synthesis of specific proteins. This means that in the overall metabolism of a cell, much less ATP is consumed than would be used if synthesis of proteins encoded in the mRNA continued long after the proteins were no longer needed.
    The short lifetime of bacterial mRNA is one criterion used to identify mRNA in bacteria. A common experimental technique to determine whether a particular RNA molecule or class of RNA molecules is mRNA is the pulse-chase experiment. RNA is labeled briefly by growing bacteria in the presence of a radioactive precursor such as [3H]uridine. Then the bacteria are switched to a medium containing no [3H]uridine and a high concentration of nonradioactive urdine and samples are removed at specific times for analyses. The RNA is isolated and different species are separated by gel electrophoresis or centrifugation and detected by their radioactivity. A stable radioactive RNA molecule will be present through many generations, whereas a radioactive mRNA molecule will decrease with a half-life of two to three minutes. One difficulty with this technique is that bacteria contain some long-lived mRNA molecules and these would be misclassified. A b......

Bacterial mRNA

Posted by star on 2018-10-25 19:13:11

    Our examination of the regulation of mRNA synthesis begins by considering some characteristics of mRNA, the sequence of which is determined by a specific template DNA sequence within the bacterial chromosome. Because transcription proceeds in a 5'→3'direction and transcription and translation occur in the same cellular compartment, the bacterial protein synthetic machinery can start to read the 5'-end of mRNA before the 3'-end is formed. Therefore, a bacterial cell does not have a chance to alter a nascent mRNA molecule before the protein synthetic machinery begins to translate it. The situation is different in eukaryotes. Because transcription occurs in the cell nucleus and translation occurs in the cytoplasm, the eukaryotic cell can convert the primary transcript to mature mRNA in the cell nucleus before the mRNA is required to direct protein synthesis in the cytoplasm.
    Protein synthetic machinery leads the mRNA nucleotide sequence in groups of three bases or codons. Each codon specifies an amino acid or a termination signal. The protein synthetic machinery begins polypeptide synthesis at a start codon located toward the5'_end of the mRNA and continues synthesis in a 5'→3'direction until it encounters a termination codon. The segment of mRNA that codes for a polypeptide chain is called an open reading frame (ORF) because the protein synthetic machinery begins reading the segment at a specific start codon and stops reading it at a specific start codon and stops reads it at a specific termination codon. A DNA segment corresponding to an open reading frame plus the translational start and stop signals for protein synthesis is called a cistron and an mRNA encoding a single polypeptide is called monocistronic mRNA. Although the terms cistron and gene are sometimes used interchangeably to describe bacterial DNA segments that specify polypeptides, the term gene has a broader meaning because it also includes the prom......

RNG chains in bacterial transcription

Posted by star on 2018-10-24 18:58:13

    RNA polymerase is a highly processive macromolecular machine. Structural factors that help to maintain the transcription elongation complex as an intact unit when it is paused include: the sliding clamp that binds the DNA template in front of the core RNA polymerase; The RNA exit tunnel that holds the nascent RNA and the segment of nascent RNA that is held to the DNA template by hydrogen bonds. Specific mechanisms are required to release RNA polymerase from the TEC. Two transcription termination pathways, intrinsic termination and Rho-dependent termination, contribute about equally to this release in E. coli.
    The intrinsic termination pathway is so named because it takes advantage of the core RNA polymerase's intrinsic catalytic activity to terminate transcription. Although nucleotide sequences specifying intrinsic terminators are present on DNA, it is actually the nascent, RNA (and not DNA) that triggers the transcription termination response. Two sequence motifs on the nascent RNA strand are essential for intrinsic terminator function.
    The first motif, a G-C rich inverted repeat, allow the RNA to fold into a stem and loop structure that reaches to within seven to nine nucleotides of the 3'-end of the nascent RNA strand. Mutations that maintain the stable stem structure are usually tolerated. Those that decrease the stem structure's stability tend to reduce or eliminate termination. Multiple mutations within the stem region also may lead to loss of terminator activity even though the stem structure retains its stability. Therefore, secondary RNA structure is important in determining intrinsic terminator activity but nucleotide sequence within the stem also appears to make a contribution.
    The second motif, a run of eight to ten nucleotides that consists mostly of uridines, comes immediately after the stem and loop structure. Intrinsic terminators appear to act by first caus......

The transcriptional elongation complex

Posted by star on 2018-10-24 18:51:39

    As the initiation stage comes to an end, the flexible flap closes to generate a transcription elongation complex (TEC), consisting of core RNA polymerase, template DNA, and a growing RNA chain elongation involves a catalytic cycle in which:
1. The nucleoside triphosphate moves through the secondary channel to reach the binding site. The passage of nucleoside triphosphates through the secondary channel may limit the elongation rate because there is only a one in four chance that the correct nucleotide will move through the secondary channel and reach the binding site.
2. A pair of electrons on the 3'-hydroxyl at the growing end of the RNA strand displaces the pyrophosphate group from the NTP to form the 5' to 3' phosphodiester bond.
3. The core RNA polymerase moves one nucleotide downstream. RNA polymerase moves along the DNA template at about 30 nucleotides per second. The incoming nucleoside triphosphates provide sufficient energy to synthesize the phosphodiester bond and drive the RNA core polymerase one nucleotide downstream.
    The DNase footprint of the transcription elongation complex is about 35 bp shorter than that of the initiating complex. Nevertheless, transcription elongation complex is the more stable of the two complexes. Approximately 14 bp within the region protected from DNase are melted, forming a transcription bubble. The first eight nucleotides within this bubble are paired with the RNA chain.
    The transcription bubble size appears to remain the same during the elongation process as RNA polymerase moves downstream because double-stranded DNA opens in front of the bubble and re-forms behind it. Conventions for numbering nucleotides in the transcription elongation complex are as follows: The entry position for the incoming nucleotide is + 1 and nucleotides downstream from this position (nucleotides in front of moving RNA polymerase) are +2, +3, and so forth. The 3'-ter......

    Although crystal structures provide a great deal of information about the way that RNA polymerase initiates transcription, additional types of experimental information are required to explain how the active center in RNA polymerase moves during abortive initiation and how the system acquires the energy needed for RNA polymerase to escape the promoter.
    Two experiments performed to examine how the active center moves during abortive transcription initiation appeared to provide contradictory observations. First, the observation that abortive initiation leads to the formation of RNA products up to-8-10 nucleotides in length suggests that the RNA polymerase active center moves relative to the DNA that it acts on. Yet in apparent contrast, DNA-footprinting experiments indicate that the enzyme does not move during abortive transcription because it protects the same up-stream DNA fragment before and after abortive RNA synthesis.
    Three models have been offered to reconcile these seemingly contradictory observations. The scrunching model proposes that DNA is pulled into the RNA polymerase holoenzyme as the DNA unwinds to form the open complex. The energy stored in the scrunched DNA is then used for promoter escape. The inchworm model proposes that the leading edge of RNA polymerase advances during the early stage of transcription initiation to allow the active center to move forward, while the other end of the enzyme remains anchored to the upstream DNA region. The energy stored in the stretched protein is used for promoter escape. Finally, the transient excursion model proposes transient cycles of forward enzyme motion during abortive RNA synthesis and backward enzyme movement after abortive transcript release with long intervals between cycles. The long interval times between cycles was postulated to explain the footprinting observations.
    Richard H. Ebright and coworkers performed ......

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