Productive and abortive initiation of transcription in vitro at the lac UV5 promoter

Gralla, Jay D.; Carpousis, Agamemnon J.; Stefano, James E.

doi:10.1021/bi00566a031

Cited by 70 publications

(55 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have also not observed the BMV replicase to initiate minus-strand RNA synthesis from templates with a 3Ј GGA substitution (15), as was reported with the TCV RdRp (31). Abortive initiation takes place with both DNA-dependent and RNA-dependent RNA polymerases in vitro (30,67,71). In the T7 RNA polymerase, it is part of a regulatory step that allows the polymerase to commit to the template for elongative RNA synthesis (65).…”

Section: Discussionmentioning

confidence: 99%

Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus

Hema

Gopinath

Kao

2005

J Virol

View full text Add to dashboard Cite

The 3 portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3 CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3 sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.The 3Ј ends of viral RNAs are required for the proper translation, stability, and replication of the RNAs. For RNA replication, the end of the RNA must be unwound and recognized by the viral replication machinery (7). Consistent with this need, several RNA-dependent RNA polymerases (RdRps) have a narrow template channel that can only accommodate single-stranded RNA or have mechanisms that discriminate against the use of double-stranded templates for de novo initiation (10,43,53). A potential cost of having a single-stranded 3Ј sequence is increased susceptibility to cellular nucleases. It is therefore to be expected that RNA viruses have mechanisms to protect the ends from degradation and/or to repair the end sequences.Strategies that could prevent degradation include the formation of base paired structures that can be opened through alternative base pairing, as in carmovirus (51), the binding of cellular proteins, as in phage Q␤ (5), or covalent linkage of viral proteins to the ends of viral genomes, as in picornaviruses and some DNA viruses (60). Several viral RNA repair processes have also been identified. The 3Ј ends of viral RNA genomes and their associated satellite (sat) RNAs can be repaired either by viral polymerase, by RNA recombination, or by a host-terminal transferase, including the poly(A) polymerase complex (9,11,12,19,20,28,31,47,48,61). Minus-strand viruses such as Hantaan virus (29) and Respiratory Syncytial virus (41) have ends with short repeats that apparently allow initiation of RNA synthesis within an internal repeat and then realign the nascent RNA at the end of the genome, thereby regenerating the ends of the RNAs. Other strategies may include the use of abortive initiation products or the synthesis of initiation products from mutated initiation sequence to prime the synthesis of the turnip crinkle virus (TCV) satellite RNA (10,47).A number of plant-infecting RNA viruses...

show abstract

Section: Discussionmentioning

confidence: 99%

Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus

Hema

Gopinath

Kao

2005

J Virol

View full text Add to dashboard Cite

show abstract

“…The template strand of the -10 element enters the active site of the polymerase (formed at the intersection between the large ␤ and ␤= subunits) and primer-independent RNA synthesis initiates. Often after several abortive initiation events that produce RNA transcripts 2-15 nucleotides in length (Gralla et al 1980;Goldman et al 2009), RNAP holo-enzyme escapes the promoter, dissociates, and the elongation phase of transcription ensues (Fig. 2, step 6).…”

Section: The Transcription Processmentioning

confidence: 99%

The essential activities of the bacterial sigma factor

et al. 2017

View full text Add to dashboard Cite

Abstract:Transcription is the first and most heavily regulated step in gene expression. Sigma () factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the 70 family of proteins and the essential roles they play in the transcription process.Key words: transcription, bacteria, RNA polymerase, sigma factor, gene expression.

show abstract

“…In general, transcription complexes stalled near the promoter (within the first 8 -10 bases) are much less stable than those stalled very distant from the promoter (1)(2)(3)(4). The transition between initiation and elongation is a complex process, involving release of stable promoter contacts and rearrangement of the protein and the nucleic acid (5-7).…”

mentioning

confidence: 99%

“…The enzyme is highly specific for a small 22-bp promoter and exhibits all the basic steps characteristic of the more complex multisubunit RNA polymerases (8,9). As with the latter, initially transcribing complexes, with transcripts up to about 8 nucleotides, are particularly unstable and abortive products are released with frequency (1,2,10). Recent structural and biochemical studies indicate that a dramatic transition occurs on translocation beyond position ϩ8 and, while the enzyme-DNA-RNA ternary complex is relatively unstable before the transition, the complex becomes much more stable as the complex enters into the elongation stage (5)(6)(7)(11)(12)(13)(14).…”

mentioning

confidence: 99%

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

Zhou

Martin

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.The process of transcription is characterized by distinct phases: initiation, elongation, and termination, at the simplest. In general, transcription complexes stalled near the promoter (within the first 8 -10 bases) are much less stable than those stalled very distant from the promoter (1-4). The transition between initiation and elongation is a complex process, involving release of stable promoter contacts and rearrangement of the protein and the nucleic acid (5-7). Consequently, it is not surprising that such complexes are relatively unstable. Since the released transcripts are relatively short, there should not be a large cellular price to inefficiency at this stage. In contrast, elongation complexes distant from the promoter must be very stable to productively synthesize long transcripts.The single subunit RNA polymerase from bacteriophage T7 presents an ideal model system in which to study transcription fundamentals. The enzyme is highly specific for a small 22-bp promoter and exhibits all the basic steps characteristic of the more complex multisubunit RNA polymerases (8, 9). As with the latter, initially transcribing complexes, with transcripts up to about 8 nucleotides, are particularly unstable and abortive products are released with frequency (1, 2, 10). Recent structural and biochemical studies indicate that a dramatic transition occurs on translocation beyond position ϩ8 and, while the enzyme-DNA-RNA ternary complex is relatively unstable before the transition, the complex becomes much more stable as the complex enters into the elongation stage (5)(6)(7)(11)(12)(13)(14).A previous study found that the apparent stability of complexes stepping away from the promoter follows an unexpected pattern: complexes stalled at positions ϩ10 and ϩ14 were observed to be more stable than complexes stalled at position ϩ22 (15). This result implied special properties for complexes recently clear of the promoter and suggested that the transition to el...

show abstract

Productive and abortive initiation of transcription in vitro at the lac UV5 promoter

Cited by 70 publications

References 18 publications

Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus

Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus

The essential activities of the bacterial sigma factor

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

Contact Info

Product

Resources

About