Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon. The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli. A 30-fold difference in readthrough of the UGA codon was observed. Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction. Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant. This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing. The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNATrP as compared with a suppressor fonn of the same tRNA. This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect.
Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.
The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C‐terminal end of the nascent peptide, which are specified by the two codons at the 5′ side of UGA. For the penultimate amino acid (‐2 location), charge and hydrophobicity are important. For the last amino acid (‐1 location), alpha‐helical, beta‐strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild‐type and suppressor forms of tRNA(Trp) (CAA). The influence of the −1 and −2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C‐terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.
Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8 -9-bp RNA: DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to ϳ20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (⌬1149 -1190) in the jaw domain of the bacterial RNA polymerase largest subunit (), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication. Cellular, multisubunit RNA polymerases (RNAPs) 1 participate in a complex cycle of conformational changes to initiate, elongate, and terminate RNA transcripts. Each step in this cycle is mediated by a network of protein-nucleic acid interactions composed of interconnected parts of RNAP that contact DNA and product RNA (1-9). During elongation, most nucleic acid contacts are made by two large subunits of similar structure and sequence in prokaryotic and eukaryotic RNAPs, called Ј and  in bacteria or RPB1 and RPB2 in eukaryotes. During initiation, these contacts are supplemented by sequence-specific DNA contacts made by auxiliary initiation factors ( in bacteria) that mediate promoter engagement.In both initiation and elongation complexes, a key component in this protein-nucleic acid interaction network occurs between ϳ20 bp of duplex DNA downstream of the polymerization site and a channel in RNAP composed of a trough formed mostly by Ј(RPB1) and a cover formed by the lobe domain of (RPB2). During promoter engagement, establishment of this contact is coupled to formation of the ϳ15 bp melted transcription bubble and insertion of the template DNA strand into the active site of RNAP (Refs. 1 and 10, and references therein). Upon promoter escape, when RNAP forms a transcription elongation complex (TEC), the downstream contact persists and participates in the response of RNAP to pause, arrest, and termination signals (11-16).The downstream DNA interaction, which stretches from the position of duplex melting 1-3 nt in front of the catalytic center to ϳ20 bp further downstream, can be subdivided into activesite proximal and active-site distal sets of contacts (Fig. 1, B and C). The active-site proximal set of contacts is made at ϩ5 to ϩ8 by the lobe and domain called the clamp (formed mostly by Ј), both of which can move relative to the central core of the enzyme (2, 8, 9). The active-site distal set of contacts ...
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