Genes and Heredity

DNA Replication and Gene Expression

DNA Replication

Bacterial DNA replication is accomplished by unwinding the double helix so that new DNA can be assembled by DNA polymerase. This newly synthesized strand is called the daughter strand.

Building a strand of deoxyribonucleic acid (DNA) requires each nucleotide joining another in sequence. The sugar of each nucleotide binds to the phosphate of the previous nucleotide. At one end of the DNA strand, called the 3′ (three prime) end, the nucleotide has an unbound sugar group. At the other end of the DNA strand, called the 5′ (five prime) end, the nucleotide has an unbound phosphate group. Because of the way the sugar phosphate backbone of DNA is joined, each end of the DNA strand is different. Nucleotides can be attached only to the 3′ end. When the double-helix structure forms, each strand is running antiparallel (parallel to but moving in opposite directions) to each other.

DNA replication is the process by which a double-stranded DNA molecule is replicated, or copied, to produce two new DNA strands. In order for this to occur, the bonds between nucleotides must be broken. This opens the double helix, creating a replication fork on either side of an open bubble of separated DNA strands within which replication can occur. The location on a replicating DNA molecule where the new strands will be produced is called the replication bubble. Each strand of DNA within the replication bubble will be used as a template to create new DNA by matching the base with its complement (adenine-thymine, guanine-cytosine).

DNA polymerase is the enzyme in DNA replication that assembles the new strands of DNA from the template strands. The DNA primer is the anchor or starting point for DNA polymerase to add free nucleotides to a growing strand of DNA. DNA polymerase travels down a single DNA strand from the 3′ end toward the 5′ end. Because DNA strands are antiparallel, only one strand can be made in a continuous fashion away from the replication fork traveling 3′ to 5′. The other strand must be created in fragments, called Okazaki fragments, because the DNA polymerase must travel in the 3′ to 5′ direction, which is in the opposite direction of the continuously opening replication fork and will repeatedly run into previously synthesized double stranded segments. The strand of DNA that is formed continuously during DNA replication is the leading strand. The strand of DNA that is synthesized in short segments during DNA replication is the lagging strand.

Prokaryotes such as bacteria have a single chromosome that is a double-stranded DNA molecule in a closed, circular loop. A replication bubble opens forming two replication forks that then move in opposite directions along this circle with each strand of DNA serving as a template for DNA polymerase. New daughter strands are created along the parent DNA, resulting in two new circular chromosomes. Since these new chromosomes contain one daughter and one parent strand, this type of replication is called semiconservative.

A bacterial plasmid is a small, circular piece of bacterial DNA that replicates replicates on its own and can be transferred between cells. Replication of plasmids occurs via a process called rolling circle replication. In this form of replication, an initiation protein, RepA, binds to a section of the double-stranded DNA called Ori (so named because this region serves as the origin of replication). RepA nicks, or cuts, one of the DNA strands and remains bound to the 5′ end. The free 3′ strand serves as a primer for the host DNA polymerase to replicate the opposing unnicked, or complementary, strand. The enzyme helicase unwinds the circle as the replication is taking place and the nicked strand is displaced. After the circular DNA has been replicated, RepA seals the two ends of the displaced nicked single strand. The displaced single-stranded DNA of the plasmid is now replicated using RNA polymerase, which adds a primer, and DNA polymerase, which uses that primer as a starting point to complete replication of the DNA strand. DNA ligase seals the nick, resulting in a newly formed plasmid. In addition to bacterial plasmids, many viruses, such as the human papillomavirus, are able to use rolling circle replication to rapidly reproduce many copies of their genomes.

Archaea are prokaryotic organisms that replicate their circular DNA from a single replication origin similar to bacteria. However, through gene-sequencing studies, it was discovered that transcription, translation, and DNA replication factors in archaea are more similar to eukaryotes than prokaryotes.

DNA Replication

In DNA replication, the double helix is broken so that new daughter strands can be produced by complementation. Because DNA can only be generated in one direction, one strand is made faster than the other.

DNA Gene Expression

DNA templates are transcribed into complementary mRNA strands, which are translated into proteins.

Each gene encodes a single protein, and proteins determine cell function and structure. First, genes must be transcribed into messenger RNA (mRNA). Messenger RNA, a molecule made from a DNA template, contains the complementary gene sequence, that is, the sequence opposite to the DNA strand. The genes are transcribed into mRNA using complementary base pairing with the DNA template. mRNA is then translated into proteins. In prokaryotic cells, which lack a membrane-bound nucleus, transcription and translation occur in the cytoplasm simultaneously. However, in eukaryotic cells, transcription takes place within the nucleus, after which the mRNA is transported out to the ribosomes in the cytoplasm for translation to take place.

The means by which viral transcription or translation takes place depends on the type of virus in question. For RNA viruses, there are three types that exist: positive-stranded (+)RNA viruses, negative-stranded (-)RNA viruses, and retroviruses. Positive-stranded RNA viruses are called positive because when they enter a host cell, their RNA can immediately be translated (using the host's ribosomes) into protein. Negative-stranded RNA viruses cannot have their RNA translated into proteins immediately upon host cell infection. Instead, they employ the enzyme RNA-dependent RNA polymerase to first transcribe the negative strand to a positive one. Then translation can ensue. Retroviruses are so named because they perform reverse transcription, a process of using an RNA template to create a complementary strand of DNA. This is a reversal of the first step of the central dogma. They do this by using the enzyme reverse transcriptase. Human immunodeficiency virus (HIV) is an example of a retrovirus. DNA viruses contain both a positive and a negative DNA strand. It is the positive strand that must first be transcribed into mRNA within the host cell. Then the viruses use the host cell's ribosomes to translate the mRNA into protein.

Transcription

Genes encoded on DNA must be transcribed into messenger RNA (mRNA) before proteins can be produced. A regulator gene codes for functional proteins that are used for a specific functions in the cell. A structural gene is responsible for the coding of structural proteins, those that serve as structural support within a cell. Transcription is the formation of mRNA from the template DNA strand to be used to build proteins. During transcription, the double helix of DNA must be opened around the gene that is meant to be expressed so that the mRNA can be generated. A promoter is the sequence of DNA to which RNA polymerase binds to initiate the process of transcription. Once the DNA double helix is unwound, the RNA polymerase binds to the transcription initiation site and synthesizes a mRNA strand with nucleotides that are complementary to the DNA template strand. Binding nucleotides to the growing RNA strand requires energy from adenosine triphosphate (ATP). RNA polymerase travels along the DNA template strand, building mRNA until a termination signal is reached and the transcript mRNA strand is released.

Process of Transcription

RNA polymerase builds the new mRNA strand by complementary base pairing with the template DNA strand.
Several differences exist in the transcription processes of eukaryotes and prokaryotes. In eukaryotes, transcription takes place within the nucleus and then mRNA is transported to the ribosomes in the cytoplasm for translation to take place. In prokaryotes, transcription and translation take place simultaneously within the cytoplasm. Both eukaryotes and prokaryotes use specific gene sequences called promoters, to initiate the transcriptional process and use an RNA polymerases to synthesize mRNA. Eukaryotes form an initiation complex with transcription factor proteins that control the rate of the process. Posttranscriptional modifications may occur after the mRNA is synthesized, such as capping where molecules are added to the 5′ end, polyadenylation where a chain of adenine nucleotides are added to the 3′ end, and splicing where the mRNA is cut into pieces and reassembled. Such modifications rarely occur in prokaryotes. Eukaryotic mRNA is monocistronic, meaning that the sequence codes for only one polypeptide, or protein. Prokaryotic mRNA is polycistronic, meaning the DNA coding sequence involves many different genes and the transcription product forms many proteins. Transcription is terminated, or ended, by a terminator DNA sequence, or by rho-dependent and rho-independent mechanisms. rho-dependent refers to a protein called rho that destabilizes the mRNA–DNA hybrid and rho-independent involves the newly synthesized mRNA forming a hairpin where it folds back and binds to itself.

Transcription in Eukaryotic and Prokaryotic Organisms

Eukaryotic Prokaryotic
Number of promoters Thousands to tens of thousands Three
Number of RNA polymerases Three One
Formation of initiation complex Forms initiation complex with transcription factors Does not for a complex
Sequence of transcription and translation Transcription takes place first in the nucleus and translation occurs second in the cytoplasm Transcription and translation occur simultaneously
Modification of mRNA Posttranscriptional modifications such as capping, polyadenylation, and splicing Rarely, posttranscriptional modifications such as capping
Introns present Yes No
Termination of transcription Poly-A signal and downstream terminator sequence Rho-dependent and rho-independent mechanisms

Transcription in prokaryotes and eukaryotes follows the same basic process, but there are many variations that differentiate the process in the two groups of organisms.

In eukaryotic cells, mRNA must be additionally processed before it can be exported from the nucleus. Genes are broken into segments called exons and introns. The exon is the DNA sequence within a gene sequence that codes for a specific protein; exons must be combined during mRNA processing. The intron is the DNA sequence within a gene sequence that does not code for a specific protein; introns must be removed from the mRNA during processing. Exon and introns are not present in prokaryotes. Because prokaryotes lack of a nucleus, transcription occurs within the cytoplasm.

Prokaryote versus Eukaryote Genes

Genes consist of a regulatory region at the 5′ end, a coding region, and a termination region at the 3′ end. Prokaryotic genes have a continuous coding region. In eukaryotic genes, introns must be removed and exons combined to form mRNA that codes for proteins.

Translation

Translation is the assembly of amino acids into proteins in the ribosomes through the reading of mRNA by tRNA and the ribosome. This requires three different types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Transfer RNA is the molecule that carries each amino acid to the strand of mRNA during translation of protein synthesis. The mRNA transcript is the result of translation and contains the information required to guide protein synthesis. One side of a tRNA has an three nucleotide long RNA sequence that binds to the mRNA and the other side binds an amino acid that is added to a growing polypeptide. The ribosomal RNA is the component of ribosomes that catalyzes peptide bond formation and is where protein translation takes place.

Translation begins when the ribosome assembles around the mRNA strand. The ribosome has three sites for the binding of tRNA to the mRNA. One end of the tRNA contains an anticodon that is used to temporarily bind to the mRNA strand. The anticodon is a three-nucleotide sequence that corresponds to a complementary codon in mRNA. A codon is a series of three nucleotides on an mRNA strand that codes for a particular amino acid. Every protein uses the same start codon (AUG, which encodes the amino acid methionine).

The other end of the tRNA carries the amino acid for the growing protein. The ribosome slides down the mRNA strand one codon at a time. Using the anticodon, tRNA binds to the complementary codon on the mRNA. There are two tRNA present in the ribosome during this process, bringing their amino acids close to each other. There are 20 different amino acids utilized to build proteins. Since mRNA has four different nucleotides, and three nucleotides are read for each codon, there are 64 possible codons. Because the number of possible codons exceeds the number of amino acids required, different codons can encode for the same amino acid. This redundancy helps protect against mutation because even if the gene sequence changes, the encoded protein could still be the same. For example, the amino acid phenylalanine (Phe) is specified by the codon UUU or UUC. The amino acid leucine (Leu) can be the product of any of the following codons: CUU, CUC, CUA, or CUG.

The ribosome then catalyzes the formation of a peptide bond to hold the two amino acids together. This continues until a stop codon is encountered. There are three different stop codons (UAA, UAG, UGA) that mark the end of translation. The stop codon results in the ribosome falling apart and the mRNA being released, where it can be translated again or degraded.

Translation of Proteins

Ribosomes assemble around mRNA transcripts and facilitate protein building by providing specific sites for delivery of amino acids by tRNA.
In eukaryotes, translation occurs within the rough endoplasmic reticulum (ER) or the cytoplasm. The term "rough" indicates that the ribosomes are attached to the outer cytoplasmic surface of the ER. Since prokaryotes have no organelles, both transcription and translation occur in the cytoplasm. This results in transcription and translation occurring simultaneously. Ribosomes will start forming around mRNA strands as they are being transcribed from the DNA.