[Prokaryotic Argonaute Proteins as a Tool for Biotechnology].

Programmable nucleases are the most important tool for manipulating the genes and genomes of both prokaryotes and eukaryotes. Since the end of the 20th century, many approaches were developed for specific modification of the genome. The review briefly considers the advantages and disadvantages of the main genetic editors known to date. The main attention is paid to programmable nucleases from the family of prokaryotic Argonaute proteins. Argonaute proteins can recognize and cleave DNA sequences using small complementary guide molecules and play an important role in protecting prokaryotic cells from invading DNA. Argonaute proteins have already found applications in biotechnology for targeted cleavage and detection of nucleic acids and can potentially be used for genome editing.

Prokaryotic Argonaute Proteins as a Tool for Biotechnology.

Programmable nucleases are the most important tool for manipulating the genes and genomes of both prokaryotes and eukaryotes. Since the end of the 20th century, many approaches were developed for specific modification of the genome. The review briefly considers the advantages and disadvantages of the main genetic editors known to date. The main attention is paid to programmable nucleases from the family of prokaryotic Argonaute proteins. Argonaute proteins can recognize and cleave DNA sequences using small complementary guide molecules and play an important role in protecting prokaryotic cells from invading DNA. Argonaute proteins have already found applications in biotechnology for targeted cleavage and detection of nucleic acids and can potentially be used for genome editing.

Role of Interactions of the CRE Region of Escherichia coli RNA Polymerase with Nontemplate DNA during Promoter Escape.

RNA polymerase (RNAP) recognizes promoter DNA through many interactions that determine specificity of transcription initiation. In addition to the dedicated transcription initiation σ factor in bacteria, the core enzyme of RNAP can also participate in promoter recognition. In particular, guanine residue at the +2 position (+2G) of the nontemplate DNA strand is bound in the CRE pocket formed by the RNAP ß subunit. Here, we analyzed the role of these contacts in the process of promoter escape by RNAP by studying point mutations in the ß subunit of Escherichia coli RNAP that disrupted these interactions. We found that the presence of +2G in the promoter slowed down the rate of promoter escape and increased proportion of inactive complexes. Amino acid substitutions in the CRE pocket decreased the promoter complex stability and changed the pattern of short RNA products synthesized during initiation, but did not significantly affect the rate of transition to elongation, regardless of the presence of +2G. Thus, the contacts of the CRE pocket with +2G do not make a significant contribution to the kinetics of promoter escape by RNAP, while the observed changes in the efficiency of abortive synthesis are not directly related to the rate of promoter escape.

The σ24 Subunit of Escherichia coli RNA Polymerase Can Induce Transcriptional Pausing in vitro.

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes.

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

Mechanisms of Stress Resistance and Gene Regulation in the Radioresistant Bacterium Deinococcus radiodurans.

The bacterium Deinococcus radiodurans reveals extraordinary resistance to ionizing radiation, oxidative stress, desiccation, and other damaging conditions. In this review, we consider the main molecular mechanisms underlying such resistance, including the action of specific DNA repair and antioxidation systems, and transcription regulation during the anti-stress response.

Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Characteristics of σ-dependent pausing by RNA polymerases from Escherichia coli and Thermus aquaticus.

The σ(70) subunit of RNA polymerase (RNAP) is the major transcription initiation factor in Escherichia coli. During transcription initiation, conserved region 2 of the σ(70) subunit interacts with the -10 promoter element and plays a key role in DNA melting around the starting point of transcription. During transcription elongation, the σ(70) subunit can induce pauses in RNA synthesis owing to interactions of region 2 with DNA regions similar to the -10 promoter element. We demonstrated that the major σ subunit from Thermus aquaticus (σ(A)) is also able to induce transcription pausing by T. aquaticus RNAP. However, hybrid RNAP containing the σ(A) subunit and E. coli core RNAP is unable to form pauses during elongation, while being able to recognize promoters and initiate transcription. Inability of the σ(A) subunit to induce pausing by E. coli RNAP is explained by the substitutions of non-conserved amino acids in region 2, in the subregions interacting with the RNAP core enzyme. Thus, changes in the structure of region 2 of the σ(70) subunit have stronger effects on transcription pausing than on promoter recognition, likely by weakening the interactions of the σ subunit with the core RNAP during transcription elongation.

Analysis of RNA cleavage by RNA polymerases from Escherichia coli and Deinococcus radiodurans.

RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase beta subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20 degrees C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2-3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.

Methods for selection of aptamers to protein targets.

Aptamers are synthetic single-stranded RNA or DNA molecules capable of specific binding to other target molecules. In this review, the main aptamer properties are considered and methods for selection of aptamers against various protein targets are described. Special attention is given to the methods for directed selection of aptamers, which allow one to obtain ligands with specified properties.

Differences in contacts of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter are determined by core-enzyme of RNA polymerase.

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.