Biology, asked by rehanali2392, 10 months ago

what protection does a consistent reading frame ensure? ( 250 word )​

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Answered by TopperPranshu
1

Answer:

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Answered by skchoudary
0

Answer:

An analysis of all the available sequences for prfB genes demonstrated that the frameshifting mechanism operates in many but not all bacterial species (Baranov et al., 2002). For those organisms in which the mechanism was predicted to exist, there is a strong conservation in the position of the SD-like sequence relative to the frameshift site. Closer inspection of these sequences and those of the correlating anti-SD sequences found within the 16S rRNA genes of the corresponding organism revealed that, in the majority of cases, the interaction between the SD-anti-SD sequences also encompassed the first position of the E site codon (Baranov et al., 2002), i.e., the spacing in these cases would in fact be only two, rather than the three that had been previously reported (distance to the first position of the P site codon when the UGA stop codon is at the A site). This suggested to us that the interaction between the two complementary sequences would preclude binding of the E site tRNA and might therefore facilitate its early release from the ribosome. We reasoned that the unusual situation arising from the premature release of the E site tRNA, namely a ribosome bearing only a single tRNA, the peptidyl-tRNA, at the P site, might be very unstable and thus be susceptible to slippage into the +1 frame (as illustrated in Figure 1). In order to test this hypothesis, we established an in vitro translation system capable of monitoring both the efficiency of frameshifting as well as the occupation by deacylated-tRNA at the ribosomal E site.

The SD-anti-SD interaction provokes a steric clash with codon-anticodon interaction of the E-tRNA (shaded), leading to premature release of the E-tRNA (reaction I). The two main states of the elongation cycle, the pretranslocational (PRE) and posttranslocational state (POST), are characterized and defined by the presence of two tRNAs at the A and P sites or P and E sites, respectively (for review, see Blaha and Nierhaus [2001]). Therefore, the presence of only one tRNA bound to the ribosome, in this case peptidyl-tRNALeu, induces instability in the ribosome, which in turn promotes a +1 frameshift (+1 FS) of the tRNA and enables the binding of Asp-tRNA in the new frame (as seen in reaction II). Note that the interaction between the SD-like sequence of the mRNA and the anti-SD of the 16S rRNA precludes a frameshift in the −1 direction.

An In Vitro System for Translation of the prfB Frameshift Window

An E. coli in vitro system was established that utilized an enriched synthetase fraction (see Experimental Procedures) to translate mRNAs containing the original sequence UAU-CUU-UGAC present in the prfB frameshift site (see Figure 2A). In this frameshift window, the stop codon UGA is preceded by codons for Leu (CUU) and Tyr (UAU), and the frameshift involves the slippage of the P site peptidyl-tRNALeu from the CUU codon in the +1 direction to re-base pair with UUU, allowing the decoding of the GAC in the A site by Asp-tRNAAsp. In order to test the hypothesis that the SD-anti-SD interaction causes release of the deacylated-tRNA from the E site due to a steric clash with codon-anticodon interaction of the E-tRNA-mRNA complex, we constructed two different mRNAs. The first mRNA (+SD) contained the SD-like sequence AGGGGGU found in the original prfB mRNA, spaced by two nucleotides from the first position of the P site codon CUU, and thus exhibited the predicted overlap with the first nucleotide of the E site codon UAU.

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Figure 2. The mRNAs Utilized for the Analysis of +1 Frameshifting Occurring during RF2 Synthesis

(A) mRNAs used in the in vitro translation system. Two mRNAs containing the frameshift window of RF2 mRNA, one containing the wild-type Shine-Dalgarno (SD)-like sequence (+SD) of the RF2 mRNA, and the other in which the SD sequence was removed without affecting the amino acid sequence (−SD).

(B) mRNAs used for dipeptide formation analysis. In large lettering is the unique region of each mRNA used for programming the ribosomes. The YLstop (upper) and YFstop (lower) mRNAs allow binding of the AcLeu-tRNA or AcPhe-tRNA to the P site, respectively. In each case, this places Tyr UAC and stop codon UGA in the E and A site, respectively. Only by slippage of the P site tRNAs into the +1 frame can Asp-tRNA bind to the A site and enable dipeptide formation.

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