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RNA, Messenger: RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.

Types of RNA

JoVE 10800

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use. The central dogma of molecular biology states that DNA contains the information that encodes proteins and RNA uses this information to direct protein synthesis. Different types of RNA are involved in protein synthesis. Based on whether or not they encode proteins, RNA is broadly classified as protein-coding or non-coding RNA. Messenger RNA (mRNA) is the protein-coding RNA. It consists of codons—sequences of three nucleotides that encode a specific amino acid. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNA. tRNA acts as an adaptor molecule that reads the mRNA sequence and places amino acids in the correct order in the growing polypeptide chain. rRNA and other proteins make up the ribosome—the seat of protein synthesis in the cell. During translation, ribosomes move along an mRNA strand where they stabilize the binding of tRNA molecules and catalyze the for

 Core: Biology

RNA Structure

JoVE 10799

The basic structure of RNA consists of a five-carbon sugar and one of four nitrogenous bases. Although most RNA is single-stranded, it can form complex secondary and tertiary structures. Such structures play essential roles in the regulation of transcription and translation.

There are three main types of ribonucleic acid (RNA): messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). All three RNA types consist of a single-stranded chain of nucleotides. Each nucleotide is composed of the five-carbon sugar ribose. The carbon molecules of ribose are numbered one through five. Carbon number five carries a phosphate group and carbon number one a nitrogenous base. There are four nitrogenous bases in RNA—adenine (A), guanine (G), cytosine (C), and uracil (U). Uracil is the only base in RNA that is not present in DNA, which uses thymine (T) instead. During transcription, RNA is synthesized from a DNA template based on complementary binding of the new RNA bases to the DNA bases; A binds to T, G binds to C, C binds to G, and U binds to A. Like DNA, adjacent nucleotides in RNA are linked together through phosphodiester bonds. These bonds form between the phosphate group of one nucleotide and a hydroxyl (–OH) group on the ribose of the adjacent nucleotide. This structure lends RNA its directionality—that is, the two ends

 Core: Biology

RNA Interference

JoVE 10804

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the expression of a gene by binding to its messenger RNA (mRNA) transcript, preventing the protein from being translated.

This process occurs naturally in cells, often through the activity of microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to selectively deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used to suppress genes that are overactive in diseases such as cancer. First, double-stranded RNA with a sequence complementary to the targeted gene is synthesized. Different types of double-stranded RNA can be used, including short interfering RNA (siRNA) and small hairpin RNA (shRNA). shRNA is one strand of RNA that is folded over—creating a double-stranded RNA with a hairpin loop on one side—and is a precursor of siRNA. The double-stranded RNA is then introduced into cells by methods such as injection or delivery by vectors, such as modified viruses. If shRNA is used, RNase enzymes in the cell, such as Dicer, cleave it down to the shorter siRNA, removing the hairpin loop. The siRNA then binds to an enzyme called Argonaute, which is part of a complex called RISC (RNA-induced silencing complex). Here, the two strands of the siRNA separate. One floats away w

 Core: Biology

RNA Analysis of Environmental Samples Using RT-PCR

JoVE 10104

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies…

 Environmental Microbiology

RNA Splicing

JoVE 10802

The process in which eukaryotic RNA is edited prior to protein translation is called splicing. It removes regions that do not code for proteins and patches the protein-coding regions together. Splicing also allows several protein variants to be expressed from a single gene and plays an essential role in development, tissue differentiation, and adaptation to environmental stress. Errors in splicing can lead to diseases such as cancer. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts designated to become mRNA are called precursor messenger RNA (pre-mRNA). The pre-mRNA is then processed to form mature mRNA that is suitable for protein translation. Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins whereas introns are the non-coding regions. RNA splicing is the process by which introns are removed and exons patched together. Splicing is mediated by the spliceosome—a complex of proteins and RNA called small nuclear ribonucleoproteins (snRNPs). The spliceosome recognizes specific nucleotide sequences at exon/intron boundaries. First, it binds to a GU-containing sequence at the 5’ end of the intron and to a branch point sequence containing an A towards the 3’ end of the intron. In a number of carefully-orches

 Core: Biology


JoVE 5548

Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed and analyzed without relying on prior available genomic information. During RNA-seq, RNA isolated from samples of interest is used to generate a DNA library, which is then amplified and sequenced. Ultimately, RNA-seq can …



JoVE 10794

Transcription is the process of synthesizing RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. Additionally, many other proteins and regulatory sequences are involved in the proper synthesis of messenger RNA (mRNA). Regulation of transcription is responsible for the differentiation of all the different types of cells and often for the proper cellular response to environmental signals. In eukaryotes, the DNA is first transcribed into a primary RNA, or pre-mRNA, that can be further processed into a mature mRNA to serve as a template for the synthesis of proteins. In prokaryotes such as bacteria, however, translation of RNA into polypeptides can begin while the transcription is still ongoing, as RNA can be quickly degraded. Transcription can also produce different kinds RNA molecules that do not code for protein, such as microRNAs, transfer RNA (tRNA), and ribosomal RNA (rRNA)—all of which contribute to protein synthesis. With few exceptions, all of the cells in the human body have the same genetic information in them, from neurons in the brain to muscle cells in the heart. So how do cells assume such diverse forms and functions? To a large extent, the answer lies in the regulation of transcription during development of the organism. Specifically, transcriptional regulation plays a central ro

 Core: Biology

Complementary DNA

JoVE 10818

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene. cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away. Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a pol

 Core: Biology

The Central Dogma

JoVE 10798

The central dogma of biology states that information encoded in the DNA is transferred to messenger RNA (mRNA), which then directs the synthesis of protein. The set of instructions that enable the mRNA nucleotide sequence to be decoded into amino acids is called the genetic code. The universal nature of this genetic code has spurred advances in scientific research, agriculture, and medicine. In the early 1900s, scientists discovered that DNA stores all the information needed for cellular functions and that proteins perform most of these functions. However, the mechanisms of converting genetic information into functional proteins remained unknown for many years. Initially, it was believed that a single gene is directly converted into its encoded protein. Two crucial discoveries in eukaryotic cells challenged this theory: First, protein production does not take place in the nucleus. Second, DNA is not present outside the nucleus. These findings sparked the search for an intermediary molecule that connects DNA with protein production. This intermediary molecule, found in both the nucleus and the cytoplasm, and associated with protein production, is RNA. During transcription, RNA is synthesized in the nucleus, using DNA as a template. The newly-synthesized RNA is similar in sequence to the DNA strand, except thymidine in DNA is replaced by uracil i

 Core: Biology


JoVE 10795

Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA) and other proteins are involved in the production of the chain of amino acids—the polypeptide. Proteins are called the “building blocks” of life because they make up the vast majority of all organisms—from muscle fibers to hairs on your head to components of your immune system—and the blueprint for each and every one of those proteins is encoded by the genes found in the DNA of every cell. The central dogma in biology dictates that genetic information is converted into functional proteins by the processes of transcription and translation. Eukaryotes have a membrane-bound nucleus where mRNA is transcribed from DNA. After transcription, mRNA is shuttled out of the nucleus to be translated into a chain of amino acids—a polypeptide—and eventually, a functional protein. This can take place in the cytoplasm or in the rough endoplasmic reticulum, where the polypeptides are further modified. By contrast, prokaryotes lack a nuclear compartment, so translation in prokaryotes takes place in the cytoplasm,

 Core: Biology
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