Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

2005 ◽  
Vol 58 (12) ◽  
pp. 851 ◽  
Author(s):  
Philip Hendry ◽  
Maxine J. McCall ◽  
Trevor J. Lockett
Keyword(s):  
Optimal Design ◽  
Base Pair ◽  
Hammerhead Ribozymes ◽  
Base Pairs ◽  
Physiological Conditions ◽  
Rna Transcripts ◽  
Cleavage Efficiency ◽  
Double Helices

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Michiko Kimoto ◽  
Rie Yamashige ◽  
Shigeyuki Yokoyama ◽  
Ichiro Hirao
Keyword(s):  
Base Pair ◽  
Pcr Amplification ◽  
Base Pairs ◽  
Dna Templates ◽  
Site Specific ◽  
Rna Molecules ◽  
Transcription System ◽  
Rna Transcripts ◽  
Pcr Method ◽  
Functional Components

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification andDsand pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modifiedPabases into RNA by T7 transcription. To prepareDs-containing DNA templates with long chains, theDs-Pxpair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containingDsat specific positions. Using theseDs-containing DNA templates and a biotin-linkedPasubstrate (Biotin-PaTP) as a modifiedPabase, 260-mer RNA transcripts containing Biotin-Paat a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining theDs-PxandDs-Papairs with modifiedPasubstrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

scholarly journals Symmetry in Nucleic-Acid Double Helices

Symmetry ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 737
Author(s):  
Udo Heinemann ◽  
Yvette Roske
Keyword(s):  
Nucleic Acid ◽  
Base Pair ◽  
Rna Structure ◽  
Test Tube ◽  
Base Pairs ◽  
Dna And Rna ◽  
Left Handed ◽  
Double Helices ◽  
Rna Duplexes ◽  
The Right

In nature and in the test tube, nucleic acids occur in many different forms. Apart from single-stranded, coiled molecules, DNA and RNA prefer to form helical arrangements, in which the bases are stacked to shield their hydrophobic surfaces and expose their polar edges. Focusing on double helices, we describe the crucial role played by symmetry in shaping DNA and RNA structure. The base pairs in nucleic-acid double helices display rotational pseudo-symmetry. In the Watson–Crick base pairs found in naturally occurring DNA and RNA duplexes, the symmetry axis lies in the base-pair plane, giving rise to two different helical grooves. In contrast, anti-Watson–Crick base pairs have a dyad axis perpendicular to the base-pair plane and identical grooves. In combination with the base-pair symmetry, the syn/anti conformation of paired nucleotides determines the parallel or antiparallel strand orientation of double helices. DNA and RNA duplexes in nature are exclusively antiparallel. Watson–Crick base-paired DNA or RNA helices display either right-handed or left-handed helical (pseudo-) symmetry. Genomic DNA is usually in the right-handed B-form, and RNA double helices adopt the right-handed A-conformation. Finally, there is a higher level of helical symmetry in superhelical DNA in which B-form double strands are intertwined in a right- or left-handed sense.

scholarly journals DNA sequence determinants of lambda repressor binding in vivo.

Genetics ◽  
1988 ◽  
Vol 118 (1) ◽  
pp. 21-29
Author(s):  
N Benson ◽  
P Sugiono ◽  
P Youderian
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Operator Function ◽  
Wild Type ◽  
Base Pairs ◽  
Lambda Repressor ◽  
Half Site ◽  
Better Than

Abstract The critical operator determinants for lambda repressor recognition have been defined by analyzing the binding of wild-type repressor to a set of mutant operators in vivo. Base pair substitutions at six positions within the lambda operator half-site impair binding severely, and define these base pairs as critical for operator function. One mutant operator binds repressor better than the consensus operator, and is a superoperator. The model proposed by M. Lewis in 1983 for the binding of lambda repressor to its operator accurately predicts the observed operator requirements for binding in vivo, with several minor exceptions. The order of affinities of the six natural lambda operators has also been determined.

scholarly journals Interplay of target site architecture and miRNA abundance determine miRNA activity and specificity

2017 ◽  
Author(s):  
Giovanna Brancati ◽  
Sarah H. Carl ◽  
Helge Großhans
Keyword(s):  
Mirna Family ◽  
Target Site ◽  
Site Quality ◽  
Seed Match ◽  
Base Pairs ◽  
Physiological Conditions ◽  
C Elegans ◽  
Mirna Seed ◽  
Mirna Abundance

ABSTRACTThe recognition that the miRNA seed sequence is a major determinant of miRNA activity has greatly advanced the ability to predict miRNA targets. However, it has remained unclear to what extent miRNAs act redundantly when they are members of the same family and thus share a common seed. Using in vivo studies in C. elegans, we uncover features that drive specific target repression by individual miRNA family members. We find that seed-distal complementarity to a specific family member promotes specificity. However, the extent and robustness of specificity are greatly increased by seed match ‘imperfections’, such as bulges and G:U wobble base pairs. Depending on the seed match architecture, specificity may be overcome by increasing the levels of a miRNA lacking seed-distal complementarity. Hence, in contrast to a binary distinction between functional and non-functional target sites, our data support a model where functionality depends on a combination of target site quality and miRNA abundance. This emphasizes the importance of studying miRNAs under physiological conditions in their endogenous contexts.

scholarly journals Switching Control of Expression of ptsG from the Mlc Regulon to the NagC Regulon

2008 ◽  
Vol 190 (13) ◽  
pp. 4677-4686 ◽  
Author(s):  
Samir El Qaidi ◽  
Jacqueline Plumbridge
Keyword(s):  
Central Region ◽  
Base Pair ◽  
Cooperative Binding ◽  
Transcriptional Repressors ◽  
Base Pairs ◽  
Different Characteristics ◽  
Site Discrimination ◽  
Control Of Expression ◽  
Operator Site

ABSTRACT The Mlc and NagC transcriptional repressors bind to similar 23-bp operators. The sequences are weakly palindromic, with just four positions totally conserved. There is no cross regulation observed between the repressors in vivo, but there are no obvious bases which could be responsible for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites, we have undertaken mutagenesis experiments to convert ptsG from a gene regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG, and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the AT base pair at position +/−11 from the center of symmetry of the operators with a GC base pair. Secondly, we changed the sequence of the CG base pairs in the central region of the operator (positions −4 to +4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition, these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some cooperative binding of Mlc to the two operators.

scholarly journals Presence of Germline and Full-Length IgA RNA Transcripts Among Peritoneal B-1 Cells

1998 ◽  
Vol 6 (1-2) ◽  
pp. 81-87 ◽  
Author(s):  
Rick De Waard ◽  
Peter M. Dammers ◽  
James W. Tung ◽  
Aaron B. Kantor ◽  
Jennifer A. Wilshire ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Cell Lineage ◽  
Cell Subset ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Physiological Conditions ◽  
Rna Transcripts ◽  
Iga Response ◽  
B Cell Subsets

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgAin vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline CαmRNA and mature CαmRNA transcripts. Germline Cαand mature Cαtranscripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-l-cell population in CD5+B-1a cells and CD5-B-1b cells, it was shown thatin vivoexpression of germline Cαand mature Cαtranscripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

scholarly journals Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

1994 ◽  
Vol 14 (3) ◽  
pp. 1709-1720 ◽  
Author(s):  
N B Pandey ◽  
A S Williams ◽  
J H Sun ◽  
V D Brown ◽  
U Bond ◽  
...  
Keyword(s):  
Base Pair ◽  
Point Mutations ◽  
Histone Mrna ◽  
Base Pairs ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Loop Sequence ◽  
Processing Signal

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

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