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1.
Arch Orthop Trauma Surg ; 143(1): 55-62, 2023 Jan.
Article in English | MEDLINE | ID: mdl-34117558

ABSTRACT

BACKGROUND: Knee osteoarthritis (OA) is a common, chronic, progressive and degenerative disease which affects patients' quality of life and may cause disability and social isolation. OA is a huge economic burden for the patient and a large strain for the whole healthcare system. Articular cartilage has a small potential to repair, with progressively more clinicians emphasizing cellular therapy. Subcutaneous fat tissue in human body is a large reservoir of mesenchymal stem cells (MSCs) and is been harvested in minimally invasive, simple procedure. The purpose of this study was to define a specific group of patients with knee osteoarthritis, who are the most likely to benefit from the treatment with intra-articular injection of an autologous adipose tissue (AAT). METHODS: From 2016 to 2018, 59 symptomatic bilateral and unilateral knee OA patients were treated with a single intra-articular (IA) injection of an autologous adipose tissue (AAT). Before the treatment and at the follow-up, the participant was asked to fulfill the Knee Injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee 2000 (IKDC 2000), The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), the Health Questionnaire EQ-5D-5L and to quantify the pain in the affected joint with a Numeric Rating Scale (NRS). Moreover, the patients were asked to: (i) assess their satisfaction with the effects of the conducted treatment: from 0 (unsatisfied) to 10 (very satisfied), (ii) describe the rehabilitation, if it was performed (supervised or individual and duration in weeks) and (iii) indicate any additional treatment applied, like IA injections of hyaluronic acid (HA) or platelet-rich plasma (PRP), knee arthroscopy, partial or total knee arthroplasty (TKA) at the follow-up. RESULTS: The mean age of 37 participants (16 males and 21 females) included into statistical analysis was 57.78 ± 7.39 years, the mean BMI was 31.30 ± 7.51. The questionnaires were fulfilled after the average follow-up time of 27 ± 6.5 months. A significant difference (p < 0.05) compared with the baseline, was observed in pain [NRS], WOMAC, KOOS index, pain, symptoms, ADL, Sport and Rec, QoL, EQ-5D-5L index. The satisfaction in the whole group was 6.16 ± 3.07. There was no significant difference between satisfied and unsatisfied patients in BMI and pain [NRS] at the baseline. 6 out of 7 patients with stage IV in K-L were unsatisfied with the effects of the treatment with AAT. DISCUSSION: The main conclusion of this study is that the patients with stage II of the knee OA with normal BMI are were most likely to benefit from IA injection of AAT, in contrast to the patients with stage IV, who will not beware not satisfied with the effectiveness of this kind of treatment. There were no adverse events reported at the donor site as well as in the treated knee joints.


Subject(s)
Osteoarthritis, Knee , Platelet-Rich Plasma , Male , Female , Humans , Adult , Osteoarthritis, Knee/surgery , Follow-Up Studies , Quality of Life , Treatment Outcome , Hyaluronic Acid/therapeutic use , Injections, Intra-Articular , Pain , Adipose Tissue
2.
Knee Surg Sports Traumatol Arthrosc ; 30(4): 1430-1435, 2022 Apr.
Article in English | MEDLINE | ID: mdl-34086095

ABSTRACT

PURPOSE: The purpose of this study was to evaluate the current status of education of polish surgeons in the subject of meniscus repair possibilities. The analysis of the possible correlations between the number of knee arthroscopy procedures performed by polish surgeons and their decision whether to remove or to repair the damaged meniscus has been performed. METHODS: Two-hundred and five registered orthopedic surgeons took part in surveys. The questionnaire contained the description of 20 patients with different types of meniscus damage and three questions concerning the experience in knee arthroscopy (two questions) and a choice of the treatment method (one question). Comparisons were made between knee arthroscopy experts (> 100 arthroscopies performed per year) and non-experts (≤ 100 cases). RESULTS: The questionnaire was completed by 194 knee surgeons from Poland with different levels in knee arthroscopy experience. For most cases, experts and non-experts agreed on the meniscus treatment method. Statistically significant differences in the recommended treatment between experts and non-experts were observed in 4 cases, where experts decided to repair the damage rather than to perform the meniscectomy. CONCLUSIONS: Meniscectomy remains a frequent orthopedic procedure, despite meniscal sparing having been advocated for several decades now and despite the existence of meniscus repair technique which gives good clinical outcomes-augmentation of the damaged meniscus with a collagen membrane. Polish surgeons still need education on the meniscus treatment possibilities. LEVEL OF EVIDENCE: V.


Subject(s)
Meniscus , Tibial Meniscus Injuries , Arthroscopy/methods , Humans , Meniscectomy , Menisci, Tibial/surgery , Tibial Meniscus Injuries/surgery
3.
Int J Mol Sci ; 22(15)2021 Jul 22.
Article in English | MEDLINE | ID: mdl-34360611

ABSTRACT

Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.


Subject(s)
Bacteria/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Nucleic Acid Conformation , RNA Folding , RNA, Bacterial/chemistry , Bacteria/growth & development , Bacteria/metabolism , Bacterial Proteins/genetics , Transcription, Genetic
4.
Int J Mol Sci ; 22(11)2021 May 27.
Article in English | MEDLINE | ID: mdl-34071929

ABSTRACT

Knee osteoarthritis (OA) is a degenerative knee joint disease that results from the breakdown of joint cartilage and underlying bone, affecting about 3.3% of the world's population. As OA is a multifactorial disease, the underlying pathological process is closely associated with genetic changes in articular cartilage and bone. Many studies have focused on the role of small noncoding RNAs in OA and identified numbers of microRNAs that play important roles in regulating bone and cartilage homeostasis. The connection between other types of small noncoding RNAs, especially tRNA-derived fragments and knee osteoarthritis is still elusive. The observation that there is limited information about small RNAs different than miRNAs in knee OA was very surprising to us, especially given the fact that tRNA fragments are known to participate in a plethora of human diseases and a portion of them are even more abundant than miRNAs. Inspired by these findings, in this review we have summarized the possible involvement of microRNAs and tRNA-derived fragments in the pathology of knee osteoarthritis.


Subject(s)
Biomarkers , Gene Expression Regulation , Osteoarthritis, Knee/etiology , Osteoarthritis, Knee/metabolism , RNA, Small Untranslated/genetics , Animals , Disease Susceptibility , Gene Expression Profiling , Humans , MicroRNAs , Molecular Diagnostic Techniques , Osteoarthritis, Knee/diagnosis , RNA, Transfer , Transcriptome
5.
BMC Musculoskelet Disord ; 21(1): 626, 2020 Sep 23.
Article in English | MEDLINE | ID: mdl-32967668

ABSTRACT

BACKGROUND: Meniscus repair is a challenging task in knee arthroscopy. Currently, there are a variety of arthroscopic methods available for meniscus repair. The purpose of this study was to determine a consensus in meniscus tear treatment in the environment of Polish orthopaedists. METHODS: A total of 205 registered orthopaedic surgeons participated in the surveys. The survey consisted of 35 questions regarding general arthroscopy and postoperative management, including physicians' level of expertise, anaesthesia, postoperative treatment, rehabilitation and procedures performed. Comparisons were made between knee arthroscopy experts (> 100 arthroscopies performed per year) and non-experts (≤ 100 cases) on aspects of patient care. RESULTS: The most important finding of this study was the agreement among almost all aspects of the knee arthroscopy approach. Consensus among Polish surgeons was noticed in choosing regional anaesthesia for knee arthroscopy, the lack of need for knee braces and knee medications, the of use of LMW heparin for thromboprophylaxis, 1-2 days of hospitalization, the recommendation of rehabilitation and the use of magnetic resonance as a diagnostic test for meniscus damage. Surgical expertise was significantly associated with the performance of meniscus suture procedures (p = 0.009). Experts recommended starting rehabilitation on the day of surgery (p = 0.007) and were more likely to use objective physical tests (p = 0.003). Non-expert surgeons recommended a longer period from meniscus suture to full-range knee motion (p = 0.001) and admitted that patient age does matter for meniscus repair qualification (p = 0.002). CONCLUSIONS: There is consensus among almost all issues of meniscus tear treatment in the environment of Polish orthopaedists; however, the issues of rehabilitation and the use of advanced meniscus repair techniques are associated with surgical expertise.


Subject(s)
Arthroscopy , Venous Thromboembolism , Anticoagulants , Humans , Knee Joint/surgery , Menisci, Tibial , Poland
6.
BMC Musculoskelet Disord ; 21(1): 314, 2020 May 20.
Article in English | MEDLINE | ID: mdl-32434498

ABSTRACT

BACKGROUND: Knee osteoarthritis (OA) is a common, chronic, progressive and degenerative disease which affects patients' quality of life and may cause disability and social isolation. OA is a huge economic burden for the patient and a large strain for the whole healthcare system. Articular cartilage has a small potential to repair, with progressively more clinicians emphasizing cellular therapy. Subcutaneous fat tissue in human body is a large reservoir of mesenchymal stem cells (MSCs) and is been harvested in minimally invasive, simple procedure. Up to date there is no prospective randomized controlled studies demonstrating effectiveness and role of adipose tissue injections in OA treatment. The purpose of this study is to assess functional and clinical changes among patients with symptomatic knee OA treated with intra-articular injections of autologous adipose tissue or platelet rich plasma (PRP) and to compare efficacy of both therapeutic methods. METHODS: This is a prospective, randomized, controlled study. Patients who meet inclusion criteria will be allocated to Fat Tissue group or PRP group randomly. Subjects will receive an intra articular injection with autologous adipose tissue and PRP respectively. Patients will be assessed five times: before treatment and 1, 3, 6 and 12 months after the treatment. The assessment consists of patient reported outcome measures (The Knee injury and Osteoarthritis Outcome Score, International Knee Documentation Committee 2000, the Western Ontario and McMaster Universities Osteoarthritis Index, the Health Questionnaire EQ- 5D- 5 L), three functional tests (The Timed Up and Go Test, The 5 Times Sit to Stand Test, The 10 m Walk Test) and Maximal Isometric Voluntary Contraction. DISCUSSION: This study protocol has several strengths and weaknesses. One of strongest point of this study is the wide, multidimensional functional assessment which will give a large amount of objective data. On the other hand, lack of blinding has to be considered as a risk of both subject and investigator bias. TRIAL REGISTRATION: name of registry: ClinicalTrials.gov, trial registration number: NCT04321629, retrospectively registered on date of registration.


Subject(s)
Adipose Tissue/transplantation , Osteoarthritis, Knee/therapy , Platelet-Rich Plasma/metabolism , Clinical Trials, Phase II as Topic , Humans , Injections, Intra-Articular , Pain Measurement , Patient Reported Outcome Measures , Prospective Studies , Quality of Life , Randomized Controlled Trials as Topic , Treatment Outcome
7.
Sci Rep ; 9(1): 18397, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31804585

ABSTRACT

In recent years, a number of small RNA molecules derived from snoRNAs have been observed. Findings concerning the functions of snoRNA-derived small RNAs (sdRNAs) in cells are limited primarily to their involvement in microRNA pathways. However, similar molecules have been observed in Saccharomyces cerevisiae, which is an organism lacking miRNA machinery. Here we examined the subcellular localization of sdRNAs in yeast. Our findings reveal that both sdRNAs and their precursors, snoRNAs, are present in the cytoplasm at levels dependent upon stress conditions. Moreover, both sdRNAs and snoRNAs may interact with translating ribosomes in a stress-dependent manner. Likely consequential to their ribosome association and protein synthesis suppression features, yeast sdRNAs may exert inhibitory activity on translation. Observed levels of sdRNAs and snoRNAs in the cytoplasm and their apparent presence in the ribosomal fractions suggest independent regulation of these molecules by yet unknown factors.


Subject(s)
Gene Expression Regulation, Fungal , Protein Biosynthesis , RNA, Small Nucleolar/genetics , Ribosomes/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Cold-Shock Response , Cytoplasm/drug effects , Cytoplasm/metabolism , Heat-Shock Response , Nucleic Acid Conformation , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Small Nucleolar/classification , RNA, Small Nucleolar/metabolism , Ribosomes/drug effects , Ribosomes/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Salinity , Sodium Chloride/pharmacology , Sorbitol/pharmacology , Stress, Physiological/genetics , Ultraviolet Rays
8.
Article in English | MEDLINE | ID: mdl-29883755

ABSTRACT

Ribosome-associated noncoding (ranc) RNAs are a novel class of short regulatory RNAs with functions and origins that have not been well studied. In this present study, we functionally characterized the molecular activity of Saccharomyces cerevisiae transfer RNA (tRNA)-derived fragments (tRFs) during protein biosynthesis. Our results indicate ribosome-associated tRFs derived from both 5' (ranc-5'-tRFs) and 3'-part of tRNAs (ranc-3'-tRFs) have regulatory roles during translation. We demonstrated five 3'-tRFs and one 5'-tRF associate with a small ribosomal subunit and aminoacyl-tRNA synthetases (aa-RSs) in yeast. Furthermore, we discovered that four yeast aa-RSs interact directly with yeast ribosomes. tRFs interactions with ribosome-associated aa-RSs correlate with impaired efficiency of tRNA aminoacylation.

9.
Acta Biochim Pol ; 63(4): 601-607, 2016.
Article in English | MEDLINE | ID: mdl-27785480

ABSTRACT

Small nucleolar RNAs (snoRNAs) are molecules located in the cell nucleolus and in Cajal bodies. Many scientific reports show that snoRNAs are not only responsible for modifications of other RNAs but also fulfill multiple other functions such as metabolic stress regulation or modulation of alternative splicing. Full-length snoRNAs as well as small RNAs derived from snoRNAs have been implied in human diseases such as cancer or Prader-Willi Syndrome. In this review we describe emerging, non-canonical roles of snoRNAs and their derivatives with the emphasis on their role in human diseases.


Subject(s)
RNA, Small Nucleolar/physiology , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression Regulation, Neoplastic , Humans , Inverted Repeat Sequences , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Prader-Willi Syndrome/genetics , Prader-Willi Syndrome/metabolism , RNA Interference , RNA Processing, Post-Transcriptional , Stress, Physiological
10.
FEMS Yeast Res ; 16(6)2016 Sep.
Article in English | MEDLINE | ID: mdl-27609601

ABSTRACT

Recently, a number of ribosome-associated non-coding RNAs (rancRNAs) have been discovered in all three domains of life. In our previous studies, we have described several types of rancRNAs in Saccharomyces cerevisiae, derived from many cellular RNAs, including mRNAs, rRNAs, tRNAs and snoRNAs. Here, we present the evidence that the tRNA fragments from simple eukaryotic organism S. cerevisiae directly bind to the ribosomes. Interestingly, rancRNA-tRFs in yeast are derived from both, 5'- and 3'-part of the tRNAs and both types of tRFs associate with the ribosomes in vitro The location of tRFs within the ribosomes is distinct from classical A- and P-tRNA binding sites. Moreover, 3'-tRFs bind to the distinct site than 5'-tRFs. These interactions are stress dependent and as a consequence, provoke regulation of protein biosynthesis. We observe strong correlation between tRF binding to the ribosomes and inhibition of protein biosynthesis in particular environmental conditions. These results implicate the existence of an ancient and conserved mechanism of translation regulation with the involvement of ribosome-associating tRNA-derived fragments.

11.
J Appl Genet ; 57(4): 531-541, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27020791

ABSTRACT

The ability to precisely regulate gene expression is one of the most important features of the living cells as it enables the adaptation and survival in different environmental conditions. The majority of regulatory mechanisms involve protein action, however, multiple genes are controlled by nucleic acids. Among RNA-based regulators, the riboswitches present a large group of specific domains within messenger RNAs able to respond to small metabolites, tRNA, secondary messengers, ions, vitamins or amino acids. A simple, accurate, and efficient mechanism of action as well as easiness in handling and engineering make the riboswitches a potent practical tool in industry, medicine, pharmacy or environmental protection. Hereby, we summarize the current achievements and challenges in designing and practical employment of the riboswitch-based tools.


Subject(s)
Gene Expression Regulation , RNA/genetics , Riboswitch , Bacteria/genetics , Biosensing Techniques , Cyclic GMP/analogs & derivatives , Cyclic GMP/chemistry , Lysine/chemistry , Protein Biosynthesis , Purines/chemistry , RNA Splicing , Thiamine Pyrophosphate/chemistry , Transcription, Genetic , Viruses/genetics
12.
FEBS Open Bio ; 6(12): 1186-1200, 2016 Dec.
Article in English | MEDLINE | ID: mdl-28203519

ABSTRACT

Short RNAs derived from the cleavage of tRNA molecules are observed in most organisms. Their occurrence seems to be induced by stress conditions, but still little is known about their biogenesis and functions. We find that the recovery of tRNA fragments depends on the RNA isolation method. Using an optimized RNA extraction protocol and northern blot hybridization technique, we show that the tRNA-derived fragments in yeast are widespread in 12 different growth conditions. We did not observe significant stress-dependent changes in the amounts of tRNA fragments pool. Instead, we show the differential processing of almost all individual tRNAs. We also provide evidence that 3'-part-derived tRNA fragments are as abundant as the 5'- one in Saccharomyces cerevisiae. The resulting set of S. cerevisiae tRNA fragments provides a robust basis for further experimental studies on biological functions of tRFs.

13.
Acta Biochim Pol ; 61(2): 211-6, 2014.
Article in English | MEDLINE | ID: mdl-24839615

ABSTRACT

Transfer RNA (tRNA) molecules are most commonly known as the molecular amino acids carriers and also because of the role they play in a protein biosynthesis process. However, tRNA biology has revealed stupendous levels of many unexpected discoveries that put a new light on tRNA function in different processes besides translation, like apoptosis or cancer development. In recent years various species of RNAs have been found differentially expressed in different types of cancer. In this review we focus our attention on tRNAs as well as on tRNA-derived small RNAs ex-translational functions in human cells in oncogenesis and oncobiology.


Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Neoplasms/genetics , RNA, Transfer/genetics , RNA, Untranslated/genetics , Amino Acyl-tRNA Synthetases/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Humans , Neoplasms/diagnosis , Neoplasms/pathology , Protein Biosynthesis , RNA, Transfer/chemistry
14.
Mol Cell ; 54(1): 147-155, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24685157

ABSTRACT

The structural and functional repertoire of small non-protein-coding RNAs (ncRNAs) is central for establishing gene regulation networks in cells and organisms. Here, we show that an mRNA-derived 18-nucleotide-long ncRNA is capable of downregulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unkown mechanism of translation regulation. Ribosome-targeted small ncRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules.


Subject(s)
Polyribosomes/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA, Small Untranslated/metabolism , Saccharomyces cerevisiae/metabolism , Adaptation, Physiological , Binding Sites , Exons , Gene Expression Regulation, Fungal , Osmotic Pressure , Polyribosomes/genetics , Protein Biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Stress, Physiological , Time Factors , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolism
15.
Nucleic Acids Res ; 40(9): 4013-24, 2012 May.
Article in English | MEDLINE | ID: mdl-22266655

ABSTRACT

The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.


Subject(s)
High-Throughput Nucleotide Sequencing , RNA Processing, Post-Transcriptional , RNA, Small Untranslated/metabolism , Software , Gene Expression Profiling , Gene Library , RNA Stability , RNA, Small Untranslated/chemistry , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Sequence Analysis, RNA
16.
Postepy Biochem ; 54(3): 251-63, 2008.
Article in Polish | MEDLINE | ID: mdl-19112824

ABSTRACT

The protein biosynthesis is a complicated process and not fully understood yet. According to smaller size and less complicated structure, understanding of prokaryotic 70S ribosomes is much more advanced. Eucaryotic 80S ribosomes are more complex and generate more difficulties in research. The morphology of 80S ribosome has been pretty well resolved and we know a lot about mechanism of functioning. Determination of the interactions between the ribosomes and the factors taking part in protein biosynthesis is still a great challenge. Dynamic changes of these interactions during particular steps of elongation cycle are quite difficult to understand. Conformational changes of the ribosome are of great functional and regulatory importance during protein biosynthesis. They are essential for the whole gene expression process. Only further research of the structure and function of the ribosome will lead us to knowledge about specificity of the mechanism of their action. In this article we present current opinions concerning structure and function of the eukaryotic ribosomes.


Subject(s)
Eukaryotic Cells/metabolism , Protein Biosynthesis/physiology , Proteins/metabolism , Ribosomes/chemistry , Ribosomes/physiology , Animals , Bacteria/genetics , Invertebrates/genetics , Models, Molecular , Plants/genetics , RNA, Ribosomal/chemistry , RNA, Transfer/chemistry , Rabbits , Rats , Translocation, Genetic/physiology
17.
Oligonucleotides ; 18(2): 175-86, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18637734

ABSTRACT

A ribosome undergoes significant conformational changes during elongation of polypeptide chain that are correlated with structural changes of rRNAs. We tested nine different antisense oligodeoxynucleotides complementary to the selected, highly conserved sequences of Lupinus luteus 26S rRNA that are engaged in the interactions with tRNA molecules. The ribosomes were converted either to pre- or to posttranslocational states, with or without prehybridized oligonucleotides, using tRNA or mini-tRNA molecules. The activity of those ribosomes was tested via the so-called binding assay. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis. In this article, we present that (i) before and after translocation, fragments of domain V between helices H70/H71 and H74/H89 do not have to interact with nucleotides 72-76 of the acceptor arm of A-site tRNA; (ii) helix H69 does not have to interact with DHU arm of tRNA in positions 25 and 26 after forming the peptide bond, but before translocation; (iii) helices H69 and H70 interact weakly with nucleotides 11, 12, 25, and 26 of A-site tRNA before forming a peptide bond in the ribosome; (iv) interactions between helices H80, H93 and single-stranded region between helices H92 and H93 and CCAend of P-site tRNA are necessary at all steps of elongation cycle; and (v) before and after translocation, helix H89 does not have to interact with nucleotides in positions 64-65 and 50-53 of A-site tRNA TPsiC arm.


Subject(s)
Oligonucleotides, Antisense/genetics , Peptide Chain Elongation, Translational/genetics , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Ribosomes/metabolism , Gene Targeting/methods , Lupinus/genetics , Lupinus/metabolism , Models, Molecular , Nucleic Acid Conformation , RNA, Plant/chemistry , RNA, Plant/genetics , RNA, Plant/metabolism , RNA, Ribosomal/genetics , RNA, Transfer, Phe/metabolism , Ribosomes/genetics , Seeds/chemistry , Structure-Activity Relationship
18.
Postepy Biochem ; 54(4): 354-61, 2008.
Article in Polish | MEDLINE | ID: mdl-19248581

ABSTRACT

In the sixties of the last (20th) century James Watson presented a two-sites model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. Twenty years later the E site, which was specific for deacylated tRNA was detected. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome simultanously during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years. It has been shown, that the E-tRNA prevents misincorporation of aminoacids and loss of reading frame. In other words, the ribosomal E site is a guard for accuracy of the polypeptide synthesized in the ribosomes.


Subject(s)
Genomic Instability/genetics , RNA, Transfer/metabolism , Ribosomes/metabolism , Animals , Binding Sites/genetics , Escherichia coli/genetics , Humans , Models, Molecular , Mutation, Missense , Ribosomal Proteins/metabolism
19.
Nucleic Acids Res ; 35(15): 5130-40, 2007.
Article in English | MEDLINE | ID: mdl-17660192

ABSTRACT

Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2'-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.


Subject(s)
Adenosine/chemistry , Peptide Chain Termination, Translational , Peptidyl Transferases/metabolism , RNA, Ribosomal, 23S/chemistry , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/enzymology , Base Sequence , Binding Sites , Hydrolysis , Molecular Sequence Data , Peptides/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Uridine/chemistry
20.
J Plant Physiol ; 164(4): 496-504, 2007 Apr.
Article in English | MEDLINE | ID: mdl-16704887

ABSTRACT

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for ribosome activity. We tested seven different deoxyoligonucleotides complementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-translocational states (with or without pre-hybridized oligonucleotides). We found a correlation between the level of oligomer hybridization and the inhibition of AA-tRNA binding. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis.


Subject(s)
Nucleic Acid Conformation , Peptide Chain Elongation, Translational , RNA, Plant/chemistry , RNA, Plant/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/metabolism , Base Sequence , Lupinus/genetics , Lupinus/metabolism , Models, Molecular , RNA, Plant/genetics , RNA, Ribosomal, 18S/genetics , RNA, Transfer, Phe/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Glycine max/genetics , Glycine max/metabolism , Thermodynamics
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