Genetic Signals and Nucleotide Sequences in Messenger RNA

Steitz, Joan A.

doi:10.1007/978-1-4684-3417-0_9

Cited by 88 publications

(32 citation statements)

References 233 publications

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“…5). A ribosome is believed to mask about 3 codons 3' to the codon that is being read (14). Thus, a ribosome stalled in region 1 at the adjacent Trp codons, or at the Arg codon immediately 3' to the Trp codons, would completely mask region 1 and prevent formation of stem and loop 1.2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

Oxender¹,

Zurawski²,

Yanofsky³

1979

Proc. Natl. Acad. Sci. U.S.A.

134

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The secondary structure of the terminated tip leader transcript from Escherichia coli was analyzed by RNase Ti partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the tip leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants. Transcription of the tryptophan (trp) operon of Escherichia coli is regulated at two sites, a promoter-operator and an attenuator.At the promoter-operator the rate of transcription initiation is regulated in response to chaqges in the intracellular level of free tryptophan (1, 2). At the attenuator, a site located within the transcribed 162-base-pair leader region that precedes the structural genes of the operon, transcription is either terminated to give a 140-residue leader transcript or allowed to proceed into the structural genes (3). Termination at the attenuator is regulated by the levels of charged and uncharged tRNATrP (4,5). Charged tRNATrP presumably is required for translation (6) of the segment of the leader transcript that codes for a 14-residue peptide containing adjacent Trp residues (7). The 3' half of the terminated tip leader transcript exhibits extensive secondary structure in vitro (8). Studies of tip leader mutants indicate that the capacity to form this secondary structure is essential for normalregulation of transcription termination at the attenuator (6, 9, 1(0). In the present study we demonstrate that the RNA region containing the adjacent Trp codons also participates in secondary structure. The various secondary structures that tip leader RNA can form help explain the physiological response ,of the operon as well as the behavior of the regulatory mutants we have studied. MATERIALS AND METHODSPreparation of Fingerprint Analysis of RNase Ti Oligonucleotides. Two-dimensional fingerprints of RNase Ti digests were prepared as described (11). RESULTSThe existence of extensive secondary structure in the 3' half of the E. coli terminated tip leader transcript is inferred from the resistance of this region to RNase T1 digestion (8). Analysis of the oligonucleotide products of RNase T1 partial digestion of tip leader RNA on denaturing gels suggested that the secondary structure consisted of two alternative stem and loop structures (8). To analyze further the extent and nature of the secondary structure of the terminated top leader transcript we used native gel electrophoresis to recover base-paired RNA fragments. We were particularly interested in determining the portion of the transcript that pairs with the Trp codon region, a segment known to be protected from RNase T1 attack (8). Fig. 1 shows an autoradiograph of RNase T1 partial digests of terminated Abbreviations: TBE, Tris/borate/EDTA; TME, Tris/MgCI2/ EDTA.

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Section: Resultsmentioning

confidence: 99%

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

Oxender¹,

Zurawski²,

Yanofsky³

1979

Proc. Natl. Acad. Sci. U.S.A.

134

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“…It is thus likely that some of the entries in our database are themselves 'false' but it is most unlikely that the number of these will be so high as to affect the significance of the effect that we observe. The issue is further clouded by the knowledge that initiation at internal ATG codons can occur in vivo (see Steitz, 1979, for a detailed discussion). It is therefore likely that some of the 'false' gene starts that seem to obey our rule are, in fact, capable of participating in translational initiation.…”

Section: Statistical Considerationsmentioning

confidence: 99%

“…In the absence of direct evidence from protein primary structure, it is often possible to make predictions about the genetic activity of an unknown DNA sequence from a study of the sequence itself (Staden, 1985). Steitz (1979) has reviewed the experiments and thinking that followed the observation by Dalgarno (1974, 1975) that the initiation codons of known genes in the Escherichia coli/coliphage system are preceded by a short sequence (SD sequence) of nucleotides that is directly complementary to some part of the -ACCUCCUUA-OH nucleotide sequence lying at the extreme 3' terminus of E. coli 16S rRNA. The present view of the mechanism of initiation of mRNA translation in prokaryotic systems is that a preliminary interaction between the 16S rRNA sequence and the mRNA 'points' a 30S ribosomal subunit towards an AUG or GUG triplet lying 5-9 nt 'downstream' (i.e.…”

Section: Introductionmentioning

confidence: 99%

“…Application of the methods outlined above to the X sequence allowed 46 ORFs to be identified, but in -50% of these cases the initiating codon could not be identified with certainty, while a further 20 ORFs that do not meet the usually accepted criteria can be distinguished in the sequence. Steitz (1979) drew attention to the likely importance of mRNA secondary structure and of protein -RNA interactions (possibly involving nucleotides lying downstream of the initiation codon as well as upstream sequences) in enhancing the specificity of the interaction and in stabilizing the ribosomemRNA-tRNA initiation complex.…”

Section: Introductionmentioning

confidence: 99%

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Messenger RNA recognition in Escherichia coli: a possible second site of interaction with 16S ribosomal RNA.

1988

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Examination of the nucleotides following the ATG or GTG initiation codons of a file of 251 genes from Escherichia coli has shown that 247 (98.4%) of them contain a sequence of at least three and 168 (66.9%) of them a sequence of at least four consecutive nucleotides that is complementary to some part of the 16 nt at the 5' terminus of the bacterial 16S rRNA. It is proposed that this sequence, which falls within the first 24 nt coding for the genetic message, might be involved in mRNA recognition through a mechanism analogous to the wellestablished 'Shine-Dalgarno' interaction with the 3' terminus of the 16S rRNA. Comparison of these data with data derived from a file of 117 'false' gene starts that have a Shine-Dalgarno-like sequence followed by a suitably spaced ATG or GTG triplet but which are believed not to lie at the beginnings of genetic messages shows the association that we have found to be statistically significant at the 99.9% level.

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“…Upon assembly of the ribosome at the initiation codon, these five nucleotides would be released from the protector stem, the RNA-DNA hybrid would re-form, and transcription would resume immediately. (We assume that the ribosome covers 13 bases beyond the P site at which the AUG codon is bound [46].) Indeed, the newly assembled ribosome would also release the four guanine residues (bases 102 to 105, Fig.…”

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confidence: 99%

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

1991

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Three different approaches were used to examine the regulatory effects of the amino acids specified by the peptide-coding region of the leader transcript of the ilvGMEDA operon of Escherichia coli K-12. Gene expression was examined in strains carrying an ilvGMED'-lac operon fusion. In one approach, auxotrophic derivatives were starved of single amino acids for brief periods, and the burst of j-galactosidase synthesis upon adding the missing amino acid was determined. Auxotrophic derivatives were also grown for brief periods with a limited supply of one amino acid (derepression experiments). Finally, prototrophic strains were grown in minimal medium supplemented with single and multiple supplements of the chosen amino acids. Although codons for arginine, serine, and proline are interspersed among the codons for the three branched-chain (regulatory) amino acids, they appeared to have no effect when added in excess to prototrophs or when supplied in restricted amounts to auxotrophs. Deletions removing the terminator stem from the leader removed all ilvspecific control, indicating that the attenuation mechanism is the sole mechanism for ilv-specific control.The specific control of expression of the ilvGMEDA operon in the enteric bacteria is characterized by repression of the operon when the three branched-chain amino acids are in ample supply but derepression when the supply of any one of the three is limited (14,30). This ilv-specific regulation appears to be exclusively a consequence of an attenuation mechanism ( Fig. 1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon. Thus, the rate at which the leader can be translated serves to sense whether the regulatory amino acid is in short or ample supply.As emphasized by Landick and Yanofsky (31), an important feature of the attenuation mechanism is the pausing of RNA polymerase at a certain site in the leader region. The pause allows time for the ribosome to initiate translation, and as translation proceeds, the base pairing in the protector (1:2 stem, Fig. 2) is progressively disrupted. Should ribosome movement be retarded, presumably by stalling at a regulatory codon, long enough for the bases in the upstream arm of the terminator (bases 157 to 164) to emerge on the nascent transcript, these bases can pair with those in the downstream arm of the protector (bases 98 to 105) in the antiterminator (2:3 stem, Fig. 3). It is assumed that, in the absence of ribosome stalling (repressing conditions), translation proceeds rapidly enough that the bases in the downstream arm of the protector will also be covered by the ribosome or otherwise be sterically prevented from antiterminator formation and the terminator will be formed instead. Altho...

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Genetic Signals and Nucleotide Sequences in Messenger RNA

Cited by 88 publications

References 233 publications

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

Messenger RNA recognition in Escherichia coli: a possible second site of interaction with 16S ribosomal RNA.

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

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