RNA and Protein Synthesis
Topics: 1. 2. 3. 4. 5.
Central Dogma of life: An Introduction RNA: A Linkage between DNA and Protein Transcription: Making an RNA Copy of a DNA Sequence Translation: Conversion of RNA Code into Protein Protein Synthesis
1. Central Dogma of life: An Introduction
Metabolic pathways include a chain of biochemical reactions which are organized in a particular way and every reaction is controlled by means of enzymes. If there is a loss of activity in a single enzyme, the whole pathway may be inactivated. One gene one enzyme hypothesis George Beadle and Edward Tatum revealed the relationship between the genes and metabolism. In their experimental analysis, X rays are used to cause mutations in strains of the mold Neurospora. Effects of these induced mutations were seen in the single genes and single enzymes in particular metabolic pathways. Both Beadle and Tatum won the Nobel Prize in the year 1958, for their famous proposal of one gene one enzyme hypothesis. The basic behind their proposal was “Chemical happening in the body are mediated by means of enzymes. Enzymes are all proteins and hence they are thus heritable traits. Hence, there should be a relationship between the gene and proteins.” X-rays induced mutations were done in Neurospora, and the resulting mutated spores were placed on a specifically prepared growth media. This growth media is supplemented with all the essential amino acids.
Cross was done between the X ray induced, mutated fungi with the normal forms. Resulting spores were again grown in a media by supplying only one of the twenty essential amino acids. If a spore is deficient (due to mutations) to synthesize a particular amino acid, such as Arg (Arginine), then it will grow only if Arginine was present in that growth medium. This clearly provides the proof that proteins were encoded in the gene of an organism. "One gene one enzyme" theory recommends that one gene codes for the production of one protein. "One gene one enzyme" has since been modified to "one gene one polypeptide" since many proteins are synthesized from more than one polypeptide. Doctrine of life The central dogma corresponds to the process of transcription of DNA to RNA to protein and is represented by four major stages. DNA Replication Replication is the process in which the DNA replicates its contained information in a process that involves a set of enzymes that work in harmony with one another. When DNA gets replicated, many different proteins function together and help to accomplish each of the following steps: DNA helicases unwound the two parental strands of the double helical DNA. Single stranded DNA binding proteins attach to the unwound strands, thus preventing them from winding back together. DNA polymerase binds easily to the individual strands which are firmly positioned, and catalyzes the elongation of the leading and lagging strands. The synthesis of leading strand runs continuously, with the help of DNA polymerase on it. In case of lagging strand, RNA primer is needed repeatedly for the synthesis of discontinuous strands termed as Okazaki fragments. DNA primase is one of the important polypeptides that function collectively to build the primer. In the final step, each newly synthesized Okazaki fragment is attached to the completed portion of the lagging strand and this reaction is catalyzed by DNA ligase.
Transcription of DNA into mRNA Transcription is the process in which one strand of DNA serves as a template for the synthesis of RNA. In this process, the non-coding sequences of base pairs (introns) are deducted from the coding sequences (exons) of a gene in order to transcribe DNA into messenger RNA (mRNA.) RNA processing and splicing In eukaryotic cells, the mRNA is processed and then it migrates from the nucleus to the cytoplasm. RNA splicing is one of the most important stages in RNA processing. In most of the genes, the DNA sequence exons (DNA coding for proteins) may be interrupted by stretches of introns (non-coding DNA).
In the first step, DNA inside the nucleus of the cell that includes all the exons and introns of the gene is transcribed into a complementary RNA copy called "nuclear RNA," or nRNA. In a second step, introns are removed from nRNA by a process called RNA splicing. The resulting edited sequence is called "messenger RNA," or mRNA. This finished mRNA leaves the nucleus and travels to the cytoplasm. Inside the cytoplasm, it encounters ribosomes which are the sites protein synthesis. Translation Translation is the process where DNA encodes for the production of amino acids and hence proteins. During translation, the newly synthesized mRNA (Messenger RNA) during transcription carries the coded information from the nucleus to the ribosomes present in the cytoplasm. This stored information is interpreted by the ribosomes and used for protein synthesis. Initiation The ribosome binds to the mRNA at the location of gene which contains the start codon (AUG) and it is recognized only by the initiator tRNA. This process is termed as ‘initiation’ and the ribosome proceeds to the ‘elongation’ phase of protein synthesis. Elongation During elongation, complexes composed of an amino acid linked to tRNA, successively bind to the appropriate codon in mRNA. This binding is achieved by the formation of complementary base pairs with the tRNA anticodon. Along the mRNA, the ribosome moves from codon to codon. Thus, the required amino acids are added one by one, thus translating into polypeptidic sequences stated by DNA and characterized by mRNA. Termination In the final stage, a release factor binds to the stop codon, terminating translation process and releasing the complete polypeptide from the ribosome. Proteins do not code for the production of nucleic acids like RNA or DNA or any other proteins. They are involved in almost all structural or enzymatic biological activities.
2. RNA: A Linkage between DNA and Protein
DNA in Viruses: In structure of viruses, the coats surrounding them act as antigens, initiating an antigenspecific antibody response. Viral vaccines work by either prompting the immune system to make antibodies or by supplying antibodies.
RNA Links the Information in DNA to amino acid sequence in Protein
Ribonucleic acid (RNA) was discovered after DNA. DNA is restricted to the nucleus in eukaryotes and in the nucleoid region of prokaryotes (with exceptional cases in chloroplasts and mitochondria). RNA occurs in the nucleus as well as in the cytoplasm and as part of the ribosomes that line the rough endoplasmic reticulum. There are three main kinds of ribonucleic acid, each of which is characterized according to the specific jobs carried out by them.
Ribosomal RNAs: Ribosomal RNAs (rRNAs) exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular structures which serve as the site of protein synthesis. Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% proteins. Thus ribosomal RNA (rRNA) is the construction site where the protein is constructed.
Messenger RNAs: Messenger RNAs (mRNAs) are the nucleic acids that "record" information from DNA in the nucleus of the cell. They carry this copied information to the ribosomes and are known as messenger RNAs (mRNA).In short, the messenger RNA (mRNA) is the blueprint for construction of a protein.
Transfer RNAs (tRNAs): The function of transfer RNAs (tRNA) is to select amino acids from the amino acid pool of the cell and delivering one by one to protein chains which are being constructed.
Thus transfer RNA (tRNA) is the means of transportation delivering the proper amino acid to the site at the right time. High levels of RNA are reported in the cells of developing embryos. In rapidly growing E. coli cells, half of the mass configures to ribosomes. In ribosomes, two-third part is made of ribosomal RNA or rRNA and the remaining one-third contains protein. In case of an infected cell RNA is synthesized from viral DNA, before the beginning of protein synthesis. There are some viruses like Tobacco Mosaic Virus (TMV) have RNA instead of DNA. If RNA extracted from a virus was injected into a host cell, the cell began to make new viruses. This gives clear evidence that RNA was involved in protein synthesis. RNA has ribose sugar instead of deoxyribose sugar. The base uracil (U) replaces thymine (T) in RNA. Most RNA is single stranded, although tRNA will form a "cloverleaf" structure due to complementary base pairing. In case of expressing the characters, RNA is more powerful then DNA since they directly code for the proteins to be synthesized. DNA has to be always dependent on RNA for its transmission of genetic characters.
3. Transcription: Making an RNA Copy of a DNA Sequence
Transcription: Transcription is the process of converting the information contained in a DNA segment into RNA. It begins with the synthesis of mRNA molecules containing nearly several hundred to several thousand ribonucleotides. The number of ribonucleotides depends upon the size of the protein to be made. In case, if the DNA were to project out into the cytoplasm where the ribosomes are located, in order to give the instructions for which proteins were to be made, then it would be more susceptible to get mutations from the mutagens. Hence DNA is secured inside. It uses the mRNA as the messenger and sends the information it wants to encode out to the ribosomes which in turn carry out the instructions in the cytoplasm. A large number of mRNA has been transcribed according to the specific genetic sequence inside the DNA. Each of the 100,000 or so proteins in the human body is synthesized from such different mRNAs. Synthesis of mRNA: A messenger RNA is synthesized in the cell nucleus by transcription of a particular segment of DNA. A small section of the DNA double helix unwinds, and the bases on the two strands are exposed. By forming hydrogen bonds with their complementary bases on DNA, RNA nucleotides (ribonucleotides) line up in the proper order. Then the nucleotides are joined together by a DNA dependent RNA polymerase enzyme, and mRNA forms. When DNA gets replicated, both the strands are copied. Here in this case, only one of the two DNA strands is transcribed into mRNA. This proves again that the RNA is a singlestranded molecule. If both the DNA strands are converted into respective mRNAs, the resulting mRNAs would be complementary to each other and there is a possibility of forming a double stranded RNA. Hence protein synthesis will not take place and the purpose of transcription will not be solved.
The DNA strand that is transcribed is called the template strand or the antisense strand while its complement is called the informational strand or the coding or sense strand. The template strand and the coding strand are complementary and also the template strand and the mRNA molecule are also complementary. This fact follows that the messenger RNA molecule produced during transcription is a copy of the DNA coding strand, only difference is Uracil in RNA instead of Thymine in DNA.
4. Translation: Conversion of RNA Code into Protein
Translation: Translation is the process of polymerization of amino acids to form a polypeptide chain. The mRNA determines the sequence of amino acids with the sequence of bases contained in it. The formation of peptide bond in between the amino acids requires energy and hence this energy is sustained inside the amino acids by the process of amino acylation of tRNA. Peptide bond formation is much easier between two such charged tRNAs.
Ribosomes which are attached to the rough Endoplasmic reticulum (ER) are the sites of protein synthesis. The ribosome is made up of two units, a smaller subunit and a larger subunit. The translation process begins when the small subunit encounters an mRNA. Inside the larger subunit, there are two sites where the succeeding amino acids bind to and from a peptide bond. Ribosome also plays its role as a catalyst in peptide bond formation.
The initiator tRNA locates the start codon region in the mRNA and directs the ribosome to bind at that site. The main phase of elongation continues where the amino acid taken by tRNA will get linked to the respective codon in mRNA by the formation of complementary base pairs with the anticodons of tRNA.
The ribosome moves to codon by codon and the amino acid are added like beads in a chain. When the ribosome comes across the stop codon, the elongation terminates and the completely synthesized polypeptide chain of amino acids is released out.
5. Protein Synthesis
Promoters and terminators: Promoters are DNA sequences that are the start signals for the transcription of mRNA. Terminators are the stop signals. mRNA molecules are long consisting of about 500- 10,000 nucleotides. Ribosomes- Site of protein synthesis: Ribosomes are the organelles present in all cells where protein synthesis takes place. Ribosomes consist of two-thirds rRNA and one-third protein. Ribosomes consist of a small 30S (in E. coli) and larger (50S) subunits. The larger subunit has two binding sites for tRNA and the smaller subunit has a single binding site for the mRNA. Each binding site consists of different lengths of rRNA. The 30S unit has 16S rRNA and 21 different proteins. The 50S subunit consists of 5S and 23S rRNA and 34 different proteins.
Transfer RNA (tRNA) is basically cloverleaf-shaped. It carries the proper amino acid to the ribosome when the codons identify them. At the top of the large loop are three bases, the anticodons, which is the complement of the codon. 61 different tRNAs are present, each having a different binding site for the amino acid and a different anticodon.
For the codon UUU, the complementary anticodon is AAA. Aminoacyl-tRNA synthetases control the amino acid linkage to the proper tRNA. Energy for binding the amino acid to tRNA comes from ATP conversion to adenosine monophosphate (AMP). Translation is the process of converting the mRNA codon sequences into an amino acid sequence. The initiator codon (AUG) codes for the amino acid N-formylmethionine (f-Met) and transcription cannot happen in absence of the AUG codon. The initiator tRNA/mRNA/small ribosomal unit is called the initiation complex. The larger subunit attaches to the initiation complex. After the initiation phase the message gets longer during the elongation phase.
New tRNAs bring their amino acids to the open binding site on the ribosome/mRNA complex, thus forming a peptide bond between the amino acids. The complex then shifts along the mRNA to the next triplet, opening the A site. The new tRNA enters at the A site. The elongation procedure continues until the proper protein is completed. When the codon in the A site is a termination codon ["stop" codon (U-A-A, U-G-A, or U-AG)], it signals the end of the process. No tRNA that is complementary to the Stop Codon is available, and so the process of building the protein stops. An enzyme called the releasing factor then frees the newly made polypeptide chain, also known as the protein, from the last tRNA. The mRNA molecule is released from the ribosome as the small and large subunits collapse. The mRNA can then be re-translated or it may be degraded, depending on how much of that particular protein is needed. All mRNA messages are eventually degraded when the protein no longer needs to be made.
Often many ribosomes will read the same message, a structure known as a polysome forms. In this way a cell may rapidly make many proteins.
Defects in protein synthesis
Mutations are sudden changes that may happen in the DNA. It can be defined better as a change in the DNA base sequence that results in a change of amino acid(s) in the polypeptide coded for by that gene. Alleles are alternate sequences of DNA bases (genes), and thus at the molecular level the products of alleles differ frequently by only a single amino acid, which can have a ripple effect on an organism by changing. Addition, deletion, or addition of nucleotides can change the polypeptide. Point mutations are the result of the substitution of a single base. Frame-shift mutations occur when the reading frame of the gene is shifted by addition or deletion of one or more bases.
Points to Remember:
Beadle and Tatum won the Nobel Prize in the year 1958, for their famous proposal of one gene one enzyme hypothesis. The central dogma corresponds to the process of transcription of DNA to RNA to protein and is represented by four major stages. Replication is the process in which the DNA replicates its contained information in a process that involves a set of enzymes that work in harmony with one another. In most of the genes, the DNA sequence exons (DNA coding for proteins) may be interrupted by stretches of introns (non-coding DNA). Ribonucleic acid (RNA) was discovered after DNA. There are three main kinds of ribonucleic acid, each of which is characterized according to the specific jobs carried out by them. Ribosomal RNAs, Messenger RNAs, and Transfer RNAs (tRNAs) Transcription is the process of converting the information contained in a DNA segment into RNA. Transcription is the process in which one strand of DNA serves as a template for the synthesis of RNA. Translation is the process of polymerization of amino acids to form a polypeptide chain. The mRNA determines the sequence of amino acids with the sequence of bases contained in it.
Translation is the process where DNA encodes for the production of amino acids and hence proteins. Ribosomes which are attached to the rough Endoplasmic reticulum (ER) are the sites of protein synthesis. Promoters are DNA sequences that are the start signals for the transcription of mRNA. Terminators are the stop signals. 61 different tRNAs are present, each having a different binding site for the amino acid and a different anticodon. The initiator codon (AUG) codes for the amino acid N-formylmethionine (f-Met). Transcription cannot happen in absence of the AUG codon. When the codon in the A site is a termination codon ["stop" codon (U-A-A, U-G-A, or U-A-G), it signals the end of the process. No tRNA that is complementary to the Stop Codon is available, and so the process of building the protein stops.