DNA polymerase then incorporates a dNMP onto the 3' end of the primer initiating leading strand synthesis. Only one primer is required for the initiation and propagation of leading strand synthesis. As the leading strand is synthesized along the lower parental strand the top parental strand becomes exposed.
The strand is then recognized by a primase which synthesizes a short RNA primer. DNA polymerase then incorporates a dNMP onto the 3" end of the primer and initiates lagging strand synthesis. The polymerase extends the primer for about 1, nucleotides until it comes in contact with the 5' end of the preceding primer. When the DNA polymerase encounters the preceding primer it dissociates. Ribonucleotides are then excised one at a time in a 5' to 3' direction. The 3' hydroxyl group on the 3' nucleotide terminus is then covalently joined, using DNA ligase, to the free 5' phosphate of the previously made lagging segment.
There are many types of DNA polymerases which can excise, fill gaps, proofread, repair and replicate. Origins: Origins are unique DNA sequences that are recognized by a protein that builds the replisome. Origins have been found in bacterial, plasmid, viral, yeast and mitochondrial DNA and have recently been discovered in mammalian DNA.
Specific origins are used for initiating DNA replication in humans. Most origins have a site that is recognized and bound by an origin-binding protein. Origin-binding Protein : binds and partially denatures the origin DNA while binding to another enzyme called helicase. Primase : synthesize the RNA primers required for initiating leading and lagging strand synthesis.
The only difference is that in RNA, all of the T nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary antisense strand. Bacteria use the same RNA polymerase to transcribe all of their genes.
During transcription, a ribonucleotide complementary to the DNA template strand is added to the growing RNA strand and a covalent phosphodiester bond is formed by dehydration synthesis between the new nucleotide and the last one added. The initiation of transcription begins at a promoter , a DNA sequence onto which the transcription machinery binds and initiates transcription.
In most cases, promoters are located just upstream of the genes they regulate. Although promoter sequences vary among bacterial genomes, a few elements are conserved.
As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it Figure 1. Figure 1. Once a gene is transcribed, the bacterial polymerase must dissociate from the DNA template and liberate the newly made RNA. This is referred to as termination of transcription.
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few significant differences see Table 1. Each transcribes a different subset of genes. Eukaryotic mRNAs are also usually monocistronic, meaning that they each encode only a single polypeptide, whereas prokaryotic mRNAs of bacteria and archaea are commonly polycistronic , meaning that they encode multiple polypeptides.
With the genes bound in a nucleus, the eukaryotic cell must transport protein-encoding RNA molecules to the cytoplasm to be translated. Protein-encoding primary transcripts , the RNA molecules directly synthesized by RNA polymerase, must undergo several processing steps to protect these RNA molecules from degradation during the time they are transferred from the nucleus to the cytoplasm and translated into a protein.
For example, eukaryotic mRNAs may last for several hours, whereas the typical prokaryotic mRNA lasts no more than 5 seconds. For example, enzymes, including those that metabolize nutrients and synthesize new cellular constituents, as well as DNA polymerases and other enzymes that make copies of DNA during cell division , are all proteins. In the simplest sense, expressing a gene means manufacturing its corresponding protein, and this multilayered process has two major steps.
The resulting mRNA is a single-stranded copy of the gene, which next must be translated into a protein molecule. Figure 1: A gene is expressed through the processes of transcription and translation. The pre-mRNA is processed to form a mature mRNA molecule that can be translated to build the protein molecule polypeptide encoded by the original gene.
Figure Detail During translation , which is the second major step in gene expression , the mRNA is "read" according to the genetic code , which relates the DNA sequence to the amino acid sequence in proteins Figure 2. Each group of three bases in mRNA constitutes a codon , and each codon specifies a particular amino acid hence, it is a triplet code. The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein. Figure 2: The amino acids specified by each mRNA codon.
Multiple codons can code for the same amino acid. The codons are written 5' to 3', as they appear in the mRNA. Figure Detail But where does translation take place within a cell? What individual substeps are a part of this process? And does translation differ between prokaryotes and eukaryotes? The answers to questions such as these reveal a great deal about the essential similarities between all species.
Within all cells, the translation machinery resides within a specialized organelle called the ribosome. In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the cytoplasm , where the ribosomes are located. On the other hand, in prokaryotic organisms, ribosomes can attach to mRNA while it is still being transcribed.
In all types of cells, the ribosome is composed of two subunits: the large 50S subunit and the small 30S subunit S, for svedberg unit, is a measure of sedimentation velocity and, therefore, mass.
Each subunit exists separately in the cytoplasm, but the two join together on the mRNA molecule. The tRNA molecules are adaptor molecules—they have one end that can read the triplet code in the mRNA through complementary base-pairing, and another end that attaches to a specific amino acid Chapeville et al.
The idea that tRNA was an adaptor molecule was first proposed by Francis Crick, co-discoverer of DNA structure, who did much of the key work in deciphering the genetic code Crick, The rRNA catalyzes the attachment of each new amino acid to the growing chain. Interestingly, not all regions of an mRNA molecule correspond to particular amino acids.
In particular, there is an area near the 5' end of the molecule that is known as the untranslated region UTR or leader sequence. This portion of mRNA is located between the first nucleotide that is transcribed and the start codon AUG of the coding region, and it does not affect the sequence of amino acids in a protein Figure 3. So, what is the purpose of the UTR? It turns out that the leader sequence is important because it contains a ribosome-binding site. A similar site in vertebrates was characterized by Marilyn Kozak and is thus known as the Kozak box.
If the leader is long, it may contain regulatory sequences, including binding sites for proteins, that can affect the stability of the mRNA or the efficiency of its translation. Figure 4: The translation initiation complex. When translation begins, the small subunit of the ribosome and an initiator tRNA molecule assemble on the mRNA transcript. The small subunit of the ribosome has three binding sites: an amino acid site A , a polypeptide site P , and an exit site E.
Here, the initiator tRNA molecule is shown binding after the small ribosomal subunit has assembled on the mRNA; the order in which this occurs is unique to prokaryotic cells. In eukaryotes, the free initiator tRNA first binds the small ribosomal subunit to form a complex.
Figure Detail Although methionine Met is the first amino acid incorporated into any new protein, it is not always the first amino acid in mature proteins—in many proteins, methionine is removed after translation.
In fact, if a large number of proteins are sequenced and compared with their known gene sequences, methionine or formylmethionine occurs at the N-terminus of all of them. However, not all amino acids are equally likely to occur second in the chain, and the second amino acid influences whether the initial methionine is enzymatically removed.
For example, many proteins begin with methionine followed by alanine. In both prokaryotes and eukaryotes, these proteins have the methionine removed, so that alanine becomes the N-terminal amino acid Table 1. However, if the second amino acid is lysine, which is also frequently the case, methionine is not removed at least in the sample proteins that have been studied thus far. Watch this video for a summary of eukaryotic transcription. What are introns and exons?
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