1 The Molecular Nature of a Gene
Walter P. Suza; Faizo Kasule; Philip W. Becraft; and Madan K. Bhattacharyya
Introduction
A gene is the fundamental unit of heredity that is inherited from a parent. The hereditary material of all living organisms is composed of DNA (deoxyribonucleic acid). The structure of DNA must allow it to store coded information that controls the biological functions of cells. DNA transmits hereditary information in a stable form by replicating accurately, ensuring that each new cell receives the same genetic instructions. Although DNA can undergo changes (we will discuss how this occurs in Chapter 3), the replication process is highly accurate, allowing reliable inheritance of genetic information. In a human cell, the total length of DNA from all chromosomes is more than 6.5 feet. DNA is tightly packaged into structures called chromosomes to fit inside the cell nucleus.
Most genes provide instructions for producing proteins involved in multiple cellular functions. The first step in the transmission of hereditary information in a gene is called transcription. Transcription involves copying the DNA nucleotide sequence into an intermediate nucleotide molecule called RNA (ribonucleic acid). For protein-coding genes, the initial RNA transcript, known as precursor mRNA (pre-mRNA), is processed into a mature messenger RNA (mRNA). The mRNA carries the information needed to synthesize a protein through a process called translation. Proteins are composed of 20 different amino acids, each with unique chemical and physical properties, linked together by peptide bonds. The order of amino acids is dictated by the sequence of nucleotide bases in the mRNA, and this amino acid sequence ultimately determines the protein’s structure and function.
Chemical Structure of DNA
Nucleotides
DNA is a polymer made of nucleotide subunits. A nucleotide consists of three chemical groups: sugar, phosphate, and a nitrogen-containing base (Figure 1). The sugar in DNA is deoxyribose, a five-carbon sugar derived from ribose (a pentose), with the hydroxyl group at the 2′ carbon replaced by a hydrogen atom. The bases (Figure 2) are of two types: purines (adenine and guanine) and pyrimidines (cytosine and thymine). When a base is attached to a sugar, a nucleoside is formed; the addition of a phosphate group to the nucleoside forms a nucleotide.
Sugar-Phosphate Bonds
Phosphate groups connect nucleotides to form a DNA strand. Phosphate groups create a linkage between the 5′ carbon of the sugar in one nucleotide and the 3′ carbon of the sugar in the next nucleotide. This type of linkage is known as a phosphodiester bond.
The carbon atoms on the deoxyribose sugar are numbered (Figure 1). The phosphate bond connects carbon #3 (the 3 prime or 3′ carbon) of one sugar to the 5′ carbon of the next. This gives a DNA strand directionality because the 5′ carbon faces one way and the 3′ carbon faces the other (Figure 3).
DNA Strands
DNA exists as a double-stranded molecule. The bases on one strand form hydrogen bonds with the bases on the other (Figure 3). This is called base pairing, which is highly specific with A, only pairing with T, and C only pairing with G. Thus, the sequences on the two DNA strands are complementary. Whenever A appears on a strand, T appears in the corresponding position of the complementary strand.
In addition to containing complementary nucleotide sequences, the DNA strands are in opposite orientations (Figure 3). The 5′ end of one strand is oriented toward the 3′ end of the other. For this reason, the strands are referred to as antiparallel.
Double Helix
The final feature of the molecular structure is that DNA assumes a helical conformation. The sugar-phosphate backbone forms the outer framework of the double helix, made of alternating deoxyribose sugars and phosphate groups (Figure 3). At the same time, the nitrogenous bases are located inside the double helix, where they pair with bases of the opposite strand.
This helical twist allows DNA to have a compact and the most stable configuration, which accommodates all the molecular units and chemical bonds that make up the DNA, while still being accessible for processes like replication and transcription.
DNA Packaging
Inside the cell nucleus, DNA is assembled, or packaged, into a material composed of DNA and protein called chromatin. Chromatin protects and regulates DNA and efficiently stores the very long DNA molecules that make up chromosomes (Figure 4) within the limited space of the nucleus.
Centromeres
The centromere (Figure 4) is a chromosomal region that controls chromosome segregation at mitosis and meiosis. Centromeres connect to microtubules of the spindle apparatus, which directs their movement to opposite poles (daughter cell nuclei) during cell division. Scientists successfully isolated these centromeric sequences from yeast and engineered brewer’s yeast (Saccharomyces cerevisiae) plasmids that could replicate like the chromosomes. Through these efforts, scientists were able to pinpoint centromeric function to a DNA stretch of about 120 bp that was resistant to DNase and bound to a single microtubule.
In the plant model species Arabidopsis thaliana, centromeres contain 178 bp satellite repeats occurring in tandem arrays ranging from 0.4–1.4 Mb. In contrast, in rice and maize, the amount of satellite DNA in centromeres is more variable and can range from 60 kb to 2 Mb on different chromosomes.
Recent studies have shown that minichromosomes, small chromosomes containing telomeres, replications, and a centromere with minimal extra genetic material, can be stably engineered to act as vectors for stacking genes, offering a powerful tool to study complex trait development in plant biotechnology and through Genetic manipulation of centromere-specific genes, e.g., CENH3 from Arabidopsis, facilitated the generation of haploid plants. This discovery may benefit plant breeders in the future by helping to speed up the generation of homozygous individuals.
Telomeres
The telomere (Figure 4) lies at the end of the chromosome and confers stability by “sealing” the end of a chromosome. Telomeres consist of a long series of short, repeated DNA sequences that occur in tandem arrays and are added to the end of the chromosome during DNA replication by an enzyme called telomerase. In most plant species, the sequence TTTAGGG constitutes a conserved telomere motif.
The knowledge about the structure and function of centromeres and telomeres has profoundly impacted molecular genetics. For example, yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) have proven useful in the physical mapping of plant genomes, requiring cloning and multiplying of large (>100 kb) DNA fragments in yeast or bacterial cells.
The rise in the use of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) systems by inducing double-strand breaks (DSBs) at any chromosome position has led to the creation of artificial chromosomes and, through this, spontaneous chromosome rearrangements, leading to genome evolution and crop domestication. Construction of artificial chromosomes for important crop species such as maize may provide novel options to improve food crops in the future.
For more information read: Construction and behavior of engineered minichromosomes in maize
Nucleosomes and Histones
DNA from higher eukaryotic organisms is about 109 to 1010 base pairs (bp). Human and maize chromosomal DNA is about 109 bp, which is equivalent to 1.8 m if all chromosomal DNA were stretched end to end in a linear manner. Yet the diameter of the nucleus is only about 4–6 μm, which makes it difficult to fit chromosomal DNA inside the cell nucleus. Fitting DNA inside the nucleus is accomplished by the ability of the DNA to assume a very condensed structure. Therefore, the organization of eukaryotic DNA into chromatin is an important aspect of DNA packaging.
The ordered coiling of chromosomal DNA around a histone protein core forms the chromatin (Figure 5). Chromatin consists of nucleosomes, representing chromosomal DNA’s association with histone proteins. A nucleosome consists of about 145–147 base pairs of DNA coiled around each histone octamer, for about two complete turns. A histone octamer consists of two copies of each core histone. The total mass of the histones in the nucleus approaches that of the DNA, with two molecules of each core histone to approximately 200 bp of DNA.
Higher Order Structures
If the higher-order chromatin structure is disrupted, electron microscopy reveals the appearance of “beads on a string” (Figure 5) with a diameter of about 10 nm. The “beads” represent the DNA wrapped around histones. Nucleosome formation results in a DNA fiber that is about 10 nm and a packaging ratio of about 7. The higher-order chromatin structure results when the 10 nm fiber is coiled into a solenoid. The result of nucleosome coiling is a chromatin fiber of 30 nm that is observed by electron microscopy.
Modifications of Chromatin Structure/Packaging
The tight packaging of DNA in chromatin must be modified to allow DNA replication and transcription. The regulation of chromatin structure is complex and not completely understood, but is partly accomplished through the chemical modification (methylation and acetylation) of histone proteins within nucleosomes (Figure 6). It is important to know that the Polycomb Repressive Complex 2 (PRC2) mediates gene silencing in plants through the trimethylation of histone H3 at Lysine 27 (H3K27me3), leading to condensation of chromatin structure to form the heterochromatin. This restricts RNA polymerase II from accessing and transcribing the genes. Histone acetylation by histone acetyltransferases (HATs) using acetyl-CoA as an acetyl donor promotes open chromatin (euchromatin) and transcription activation (Figure 6).
The enzyme DNase has been used to compare the chromatin structure of DNA regions undergoing active transcription with those less transcriptionally active. Such DNAse assays (Figure 7) may be conducted on DNA isolated from two different tissues or the same tissue at different stages of development. The idea is that the more sensitive the DNA is to DNase, the more active the transcription in the tissue from which the DNA was isolated, and vice versa.
Central Dogma in Molecular Biology
Francis Crick (Figure 8), who co-discovered the structure of DNA with James Watson, proposed this fundamental theory in molecular biology in the late 1950s. Crick proposed that genetic information in any biological system flows from DNA to RNA to proteins. This ensures that the information stored in DNA can be accurately transcribed into RNA and then translated into proteins to perform cellular functions in the cell.
Watch: This video about Central Dogma.
This is the process by which genetic information is accurately copied, providing the foundation for cell division and the development of organisms.
Semi-Conservative
In the replication process, the two DNA strands separate, each serving as a template for synthesizing complementary daughter strands (Figure 9). Therefore, the sequence of the daughter strand is determined by the sequence of the template strand. After replication, each DNA molecule is a duplex consisting of one parental (template) strand and one newly synthesized daughter (copy) strand. For this reason, it is referred to as semi-conservative replication and was first demonstrated by Matthew Meselson and Franklin Stahl in 1958. Thus, DNA replication is characterized by sequence conservation from one generation to another of the two individual strands on the parental duplex, a property that ensures the accuracy of genetic material reproduction.
In the experiment conducted in the 1950s, Mathew Meselson and Franklin Stahl showed that DNA replication is semi-conservative. They showed that by growing bacteria in a medium containing heavy nitrogen (15N), a stable isotope of 14N, which resulted in denser DNA. The bacteria were transferred to a medium containing light 14N, and their DNA was extracted and separated using a density gradient. After replication, each DNA molecule consisted of one original (heavy) strand and one newly synthesized (light) strand. This experiment confirmed the semiconservative model replication.
For more information read: The Meselson-Stahl Experiment (1957–1958), by Matthew Meselson and Franklin Stahl
Replication in Eukaryotes
DNA replication occurs in the S-phase of the cell cycle (Figure 10) to synthesize new genetic material for cell division. In Eukaryotic organisms such as maize, DNA replication occurs in the nucleus. The process is complex and involves several enzymes and proteins in three main stages: initiation, elongation, and termination (Figure 11).
During initiation, DNA bound to histones in nucleosomes is made accessible to proteins and enzymes involved in replication (Figure 11). The process begins when an enzyme called DNA helicase unwinds the double helix by breaking hydrogen bonds between the two strands. The unwinding of DNA leads to the formation of two replication forks that extend in both directions to result in what is referred to as a replication bubble.
As DNA unwinds, it is forced to rotate and supercoil. The torsional strain created by the action of DNA helicase is relieved by topoisomerases. Topoisomerases introduce temporary breaks in the strands of DNA, and this allows replication to proceed without impediment. Type I topoisomerases break one strand, while type II topoisomerases cut both strands.
In elongation, the DNA polymerase synthesizes both leading and lagging strands by adding nucleotides in the 5′ to 3′ direction. The antiparallel DNA template strands are read in the 3′ to 5′ direction. However, DNA polymerase cannot initiate synthesis on its own. It requires a short RNA primer synthesized by the primase enzyme. DNA synthesis proceeds continuously on the leading strand in the direction of the replication fork. In contrast, the lagging-strand synthesis is discontinuous, resulting in short fragments referred to as Okazaki fragments, each starting with a new primer (Figure 11).
As mentioned above, DNA replication must be accurate to ensure the genetic information is correctly passed from parent cells to their daughter cells. Therefore, DNA polymerases δ and ε proofread each base added to the nascent strand to correct errors that may occur during elongation.
DNA polymerase halts when it encounters another replication fork and replicates the template, and drops off. After elongation, the RNase H enzyme removes RNA primers, and DNA polymerase replaces the gaps with DNA. DNA ligase then joins fragments by forming phosphodiester bonds between them, completing the backbone of the new strand. Replication is terminated when two replication forks meet. However, a small portion of the lagging strand may remain unreplicated, which could lead to shortening of chromosomes. To counter this, the telomerase enzyme extends chromosome ends by adding repetitive DNA nucleotides, ensuring that essential genes are not lost during cell division.
The Polymerase Chain Reaction (PCR)
Knowledge about DNA replication in living cells led to the innovative development of the Polymerase Chain Reaction (PCR), which is a versatile technique used routinely in the laboratory to amplify specific regions of the DNA template. A detailed description of the PCR technique will be provided in Chapter 6.
Essentially, PCR requires template DNA, primers, nucleotides, and cofactors, and is conducted in a thermal cycler. The enzyme DNA polymerase uses single-stranded DNA as a template for the synthesis of new strands of DNA complementary to the template. PCR relies on short synthetic DNA fragments called primers to define the segment of the genome to be copied. Through multiple cycles of DNA synthesis (denaturation, primer annealing, and strand extension), the selected region is amplified, producing many copies of the target sequence. This process produces many copies of the sample DNA, which can be used for analyses such as screening for a particular nucleotide base sequence associated with a trait. There are various types of PCR, including real-time PCR, which allows rapid and real-time detection using fluorescent probes, and reverse transcription PCR (RT-PCR), which converts RNA into DNA for amplification, and has been crucial in gene expression studies. After performing PCR, the results can be visualized via gel electrophoresis.
Transcription and Gene Structure
The DNA sequence contains information to control all biological functions, including the manifestation of traits important to agriculture (yield, drought tolerance, disease resistance, etc.). How is the information contained in DNA sequences converted into the cellular activities necessary for plants and other organisms to function? DNA sequences direct the synthesis of other molecules that perform these cellular functions through an RNA intermediate. The process of producing RNA molecules from DNA templates is called transcription. Some RNA molecules perform cellular functions directly, while many others (messenger RNAs) are used to direct the synthesis of proteins in translation.
Gene Structure
Only specific regions of chromosomal DNA undergo transcription. A gene can be defined as the region of a chromosome that is transcribed, together with associated DNA sequences that regulate transcription (Figure 12). Gene regulation is discussed further in Chapter 2.
Promoter and Terminator
The instructions for starting and stopping transcription are located within DNA sequences. Specific nucleotide segments called promoters are recognized by RNA polymerase for RNA synthesis. The promoter is located around the first nucleotide (known as +1) that is transcribed into RNA. After the transcription of the full-length RNA strand is completed, a second segment of DNA called a terminator invokes termination of RNA synthesis and the detachment of RNA polymerases from the DNA template.
The CaMV 35S promoter from the cauliflower mosaic virus (CaMV) is commonly used in genetic engineering because it is a strong promoter that directs high transcription levels. A terminator sequence, such as the nopaline synthase (NOS), is also commonly used to mark the end of the transgene sequence for proper transcription and expression in plant cells.
Transcribed Region
This region, also known as the RNA coding region, is transcribed by RNA polymerase. The transcribed region is demarcated by promoter and terminator sequences. As described below, the transcribed region may include introns, sequences removed from the mature RNA molecule during RNA processing.
Introns and Exons
As mentioned above, and described in detail below, introns are sequences removed from transcripts during RNA processing. Sequences that are retained in mature transcripts are called exons. The corresponding stretches of DNA are typically referred to with the same terms. Introns are commonly found in genes of eukaryotes but are rare in prokaryotic organisms.
Protein Coding Region
The protein-coding region of a gene is composed of the sequence of nucleotides that code for amino acids (Figure 13). As described further in the section on translation, the coding region begins with an ATG start codon (AUG in RNA) and then ends with one of three stop codons. These sequences include only exons, but not all exonic sequences are protein-coding, as they may consist of untranslated regions.
Untranslated Regions (UTRs)
Mature transcripts contain some sequences that do not code for amino acid sequences in proteins. These are referred to as untranslated regions or UTRs. Most mRNA transcripts contain a 5′ and a 3′ UTR. The 5′ UTR contains sequences toward the 5′ end of the mRNA sequence, before the start codon. These sequences can often be important for translational regulation, and sometimes other functions. The sequences following the stop codon are the 3′ UTR. The 3′ UTR may also have important functions regulating transcript stability or directing transcript localization within cells, or sometimes even transport (trafficking) between cells.
UTRs and introns are often useful in genetic studies. Protein-coding regions are under strong selective pressure to produce functional proteins; therefore, sequence variation is relatively rare. UTRs and introns, on the other hand, are under less stringent selection and are the sources of sequence variants that can be used to develop genetic markers.
Transcription
Genetic information of DNA is transferred to an intermediate molecule called RNA, which is often translated into amino acid sequences used to build proteins. RNA is a nucleic acid, like DNA, but with some important differences. RNA contains ribose sugar, which has an OH group at the 2′ position, while deoxyribose is found in DNA, has a hydrogen at the same position. RNA molecules are primarily single-stranded, instead of being double-stranded. RNA contains a uridine (U) base and does not contain a thymidine base. The other bases (A, C, G) are contained in both RNA and DNA. U has the property of base-pairing with A.
A DNA template directs RNA synthesis in a process called transcription. A protein complex containing the enzyme RNA polymerase synthesizes an RNA molecule by adding nucleotides to the 3′ end of a growing chain. The principle of base pairing is used again, and each nucleotide base added is complementary to the corresponding base on the DNA template. Thus, the RNA is complementary in sequence to the DNA template strand, also referred to as “antisense” or “negative” strand. The RNA is identical in sequence (except U replaces T) to the other strand, which is called the “sense” or “positive” strand. Because transcription produces RNA molecules, they are often referred to as transcripts.
Transcription in eukaryotes occurs in the nucleus and is carried out by three polymerase enzymes (RNA polymerase I, II, and III). These enzymes share subunits and structural features but are known to transcribe different types of genes. RNA polymerase I transcribes genes for ribosomal RNA (rRNA), RNA polymerase II transcribes most genes, including protein-coding genes, and we shall focus on this polymerase for this section, while RNA polymerase III transcribes transfer RNA (tRNA) and other small RNAs.
Like replication, transcription in eukaryotes occurs in three stages: initiation, elongation, and termination. Eukaryotic RNA polymerase II requires multiple proteins called general transcription factors, collectively known as TFII (Transcription Factor RNA polymerase II), to assemble at the promoter region for the polymerase to start transcription during initiation. Nucleosomes are also unpacked during this stage to make DNA accessible to RNA polymerase II. With the help of several transcription factors, RNA polymerase binds to a region upstream of the gene called referred to as the promoter, often containing a TATA box, usually 23-35 base pairs before the transcription start site, with the help of the TATA-binding protein (TBP) subunit. This distorts the DNA by creating a bend, and this facilitates the binding of TFIID to the TATA box via TBP. TFIIA binds to stabilize the TFIID-TATA box interaction. Other TFIIs are recruited like TFIIA, TFIIB, TFIIF, TFIIE, TFII, and TFIIH (Figure 14).
Unlike DNA polymerase, which requires a primer to start replication, RNA polymerase does not need a primer to initiate RNA synthesis (Figure 15). During the elongation phase, once bound to the DNA, TFIIH acts as a DNA helicase to unwind the double helix to expose the template strand. This also has a kinase activity that phosphorylates the C-terminal domain (CTD) of RNA polymerase II, leading to a conformational change that signals RNA polymerase II to start elongation. Once this process begins, most TFIIs are released to make them available for new transcription rounds with a new RNA polymerase.
The RNA polymerase II moves along the DNA in the 3′ to 5′ direction, synthesizes complementary RNA in the 5′ to 3′ direction through nucleophilic attack of the 3′-OH group to the growing RNA chain on the α-phosphate of the incoming ribonucleotide triphosphate, and rewinds the DNA behind it. This produces a primary transcript called pre-messenger RNA (pre-mRNA) (Figure 15). The RNA polymerase II transcription termination signal is perceived when the enzyme transcribes 1000-2000s nucleotides beyond the gene’s end. Termination is triggered, causing RNA to be cleaved between the AAUAAA sequence and a downstream GU-rich region.
Unlike DNA replication, RNA polymerase lacks proofreading activity; however, the cell compensates through RNA processing, transcript surveillance, and various regulatory systems like selective degradation of defective RNA that ensure transcript fidelity and proper function.
RNA Processing
As described above, exons are protein-coding (translated) sequences, while introns are intervening sequences that interrupt exons. The removal of introns, along with the addition of a 5′ cap and a 3′ poly-A tail to the mRNA, is called RNA processing. A simplified illustration of transcription and RNA processing is shown in Figure 16. The detailed mechanisms of these processes are discussed below.
Intron Splicing
The process of transcription produces pre-mRNA that contains both introns and exons. The process of splicing involves the removal of introns from pre-mRNA and the joining together of the exons (Figure 17). A spliceosome, a large ribozyme complex made up of a group of RNA and proteins performs the splicing reaction on the C-terminal domain (CTD) of RNA polymerase II. In yeast, the spliceosome has about 90 proteins, while in humans 170 proteins. This complex functions with the help of small nuclear ribonucleoproteins (snRNPs), i.e., U1, U2, U4, U5, and U6, which consist of about 150 nucleotides and other secondary structures and a core of the seven Sm proteins, which form a ring and bind the conserved AAUUUGUGG Sm site.
For splicing to occur, the spliceosome recognizes three splicing signals, i.e., the 5′ splice donor site, the branch point sequence, and the 3′ splice acceptor site. The process involves several steps, and it starts in complex E, where U1 binds to the 5′ splice site, which is the commitment step that defines the intron-exon boundaries. This is followed by the binding of U2 to the branch point in complex A, which displaces the Adenosine nucleotide for nucleophilic attack. Then complex B will form when U6 replaces U1 at the 5′ splice site, which causes U4 to base pair with U6, as a regulator. This then causes U5 to align the exons. Upon U4 dissociation, this allows U6 to base pair with U2, and this allows catalysis by binding Mg 2+ ions; these stabilize the negative charge on the phosphate backbone and activate the nucleophile, i.e., the OH group, and create a catalytic center with U2 and the branch point. Two transesterification reactions follow this: First, the 2′ OH group of the branch point Adenosine nucleophilic attacks on the 5′ splice site. This cleaves the 5′ exon from the intron, forming a lariat structure where the intron will loop and connect to the branch point Adenosine via a 2′-5′ phosphodiester bond. Second, the newly freed 3′OH of the 5′ exon attacks the phosphate at the 3′ splice site. This process joins the two exons together and releases the intron.
Introns sometimes serve as boundaries for sequences encoding functional protein domains, leading to the possibility of new and variant proteins by exon shuffling. Also, introns can produce variant RNA forms through alternate splicing, allowing more than one gene product from a single gene. This is because the spliceosome commitment depends on whether exon or intron recognition sequences are disrupted, often leading to exon skipping or intron retention. It is important to note that alternative splicing is very common in eukaryotes, with 75% of human transcripts undergoing this process. This, therefore, affects protein localization, activity, and function. Some introns result from the insertion of transposable elements and may be spliced perfectly or imperfectly, offering more possibilities for new genetic diversity.
For more information read: Alternative Splicing within and between Drosophila Species, Sexes, Tissues, and Developmental Stages
For more information read: Differential isoform expression and alternative splicing in sex determination in mice
5′ Capping
Once RNA polymerase has synthesized about 25 nucleotides of RNA, the 5’ end of the new RNA is modified by the addition of a “cap” (Figure 18). The 5′ capping adds a 7-methyl guanidine to the first nucleotide of the mRNA molecule, usually an adenine or guanine. The phosphodiester linkage between 7-methyl guanidine and the target nucleotide is 5′-5′ instead of 5′-3′, and 3 phosphates rather than one are retained in the linkage. The capping process involves three enzymes acting in order: a phosphatase removes a phosphate from the 5 ′ end, a guanyl transferase adds guanosine monophosphate (GMP) in orientation, and a methyl transferase methylates the added guanosine. These enzymes are bound to the phosphorylated RNA polymerase tail, enabling rapid capping as nascent RNA is produced. The cap stabilizes the 5′ end of the mRNA, protecting it from degradation in the cytosol, and plays a role in translation initiation.
Poly Adenylation
The transcription of a gene may proceed beyond what ends up as the 3′ end of mature mRNA. Thus, the 3′ end of mRNA is formed after transcription. The enzyme poly(A) polymerase adds numerous adenosines to the 3′ end, resulting in the poly(A) tail (Figure 18). The poly(A) tail is necessary to process and transport mRNA to the cytoplasm properly. The poly(A) tail is also important for mRNA stability by protecting against exonucleolytic degradation and initiating translation in eukaryotic organisms by helping Ribosomes recognize mature mRNA.
In eukaryotes, cleavage and polyadenylation are two important processes that control the formation of the mRNA 3′ end in the nucleus. RNA polymerase II will transcribe beyond the location where the mature mRNA will terminate, about 20 nucleotides upstream of the cleavage site. This is usually a conserved polyadenylation site, often AAUAAA, followed by a pre-mRNA cleavage point downstream. Usually, a GU-rich region is downstream of this site to improve the efficiency of cleavage, and following cleavage, an enzyme known as poly(A) polymerase (PAP) adds the poly(A) tail of about 250nts to the 3′ end. The cleavage and polyadenylation involve many protein complexes which coordinate these processes; cleavage and polyadenylation specificity factor (CPSF) binds the AAUAAA signal, cleavage stimulation factor (CstF) that the GU-rich region, and cleavage factors I and II (CFI and CFII) participate in cleavage. Another protein, Poly(A) Binding Protein II (PABII), binds to the Poly(A) tail to promote tail elongation and stability. All these processes are coordinated on the C-terminal domain (CTD) of RNA polymerase II. The Poly(A) tail helps in the nuclear export of mature mRNA transcripts
For more information read: A Plant Poly(A) Polymerase Requires a Novel RNA-binding Protein for Activity
Codons and the Genetic Code
The search for the genetic code revealed that genetic information is stored in nucleotide triplets called codons. The genetic code is degenerate because many amino acids are specified by more than one codon. Sixty-one of the 64 possible combinations of the three bases in a codon are used to code for specific amino acids. Three stop codons, UAA, UAG, and UGA, do not code for amino acids but specify the termination of peptide chain synthesis during translation. The AUG start codon initiates polypeptide synthesis and codes for the amino acid methionine.
Ribosomes, tRNA, and Anti-codons
The ribosomes are a large molecular complex that assembles several ribosomal RNAs (rRNA) and many proteins. Ribosomes are the machinery of protein synthesis, facilitating the ordered addition of amino acids to a nascent polypeptide chain under the guidance of an mRNA template. Before initiating protein synthesis, the ribosome occurs in two separate subunits of size 60s and 40s (The “s” stands for Svedberg units and is a measure of a particle’s sedimentation rate during centrifugation).
The tRNA determines the meaning of a codon for a specific amino acid (transfer RNA). Each transfer RNA contains a triplet of nucleotides referred to as an anticodon, which is complementary to a specific codon. For each of the 20 amino acids, a specific enzyme
(aminoacyl-tRNA synthetase) catalyzes its linkage to the 3′ end of its specific tRNA adapter. The function of aminoacyl-tRNA synthetase is critical as it checks for accuracy in protein synthesis by adding a specific amino acid to a specific tRNA molecule. Thus, a particular amino acid is targeted to each codon triplet of mRNA.
Translation
The process of translation in eukaryotes is conserved and occurs in the cytoplasm on ribosomes with the help of several translation factors and takes place in 3 steps: initiation, elongation, and termination.
Translation initiation
A pre-initiation phase in eukaryotes starts with the charging of transfer RNA (tRNA), which involves amino acid activation using ATP. It is attached to its specific tRNA by an aminoacyl-tRNA synthetase. This forms an aminoacyl-tRNA complex. In eukaryotes, translation starts with methionine delivered by the initiator tRNA (Met-tRNAi), different from internal methionine. The 43S pre-initiation complex (43S PIC) assembles when the 40S small ribosomal subunit binds to multiple eukaryotic initiation factors (eIFs) (Figure 19).
This process starts with the binding of eIF3 to the solvent surface side of 40S, followed by the binding of eIF1 and eIF1A, which causes a confirmation change that opens the mRNA entry channel. In parallel, the eIF2 with GTP bound then binds to the Met-tRNAi, forming a ternary complex, which together with eIF5 joins the 40S to complete the 43S complex. During this, mRNA is prepared for loading by the actions of the eIF4F complex, which includes eIF4E that binds the 5′ cap, eIF4A, a helicase, and eIF4G. This scaffolding protein bridges mRNA to the ribosome and interacts with Poly(A) binding protein to circularize the mRNA. The resultant mRNA-eIF4F complex is recruited to the 43S complex, forming the 48S pre-initiation complex.
During Initiation, the 48S complex scans the mRNA in the 5′ to 3′ direction until the correct start codon is located, usually AUG with a favorable Kozak context, i.e., with A or G three nucleotides before the AUG (position –3) and a G immediately after the AUG (position +4). The complex of eIF1 and eIF1A maintains the ribosome in an open conformation of the 40S subunit and ensures the correct start codon is recognized. Once the correct AUG is located, GTP-bound to eIF2 is hydrolyzed by eIF5B to promote eIF1 dissociation, allowing the 60S ribosomal subunit to join the 48S complex, and collectively they form the functional 80S initiation complex, with Met-tRNAi Met correctly positioned in the P site, ready to start the elongation phase or peptide chain formation.
Peptide Chain Formation
Elongation in eukaryotic translation begins once the initiator Met-tRNA is correctly positioned in the P site of the assembled 80S ribosome, a structure necessary to align each successive aminoacyl-charged tRNA to transfer its amino acid to the growing polypeptide. Successive amino acids are attached to the growing polypeptide by the formation of peptide bonds that form between the carboxyl group of one amino acid and the amino group of the next. Elongation has three main steps: (1) decoding, (2) peptide bond formation, and (3) translocation.
Aminoacyl-tRNAs (aa-tRNAs) enter the A site of the ribosome with the help of GTP-bound elongation factor eEF1A. GTP is hydrolyzed when the correct codon-anticodon pairing is formed, and this releases eEF1A and accommodates aa-tRNA into the A site. A guanine nucleotide exchange factor, eEF1B, recycles eEF1A by exchanging GDP for GTP. The amino acid in the A site forms a peptide bond with the growing polypeptide chain in the P site, and the peptidyl transferase center of the 60S ribosomal RNA catalyzes this. Finally, translocation occurs when the GTPase eEF2 binds the ribosome and hydrolyzes GTP, shifting peptidyl-tRNA into the P site and deacylated tRNA to the E site, where it exits.
Translation elongation proceeds codon by codon in the 5′ to 3′ direction along mRNA, synthesizing the polypeptide chain from the amino (N-) terminus to the carboxy (C-) terminus. Like nucleic acids, polypeptides also have molecular directionality. One end of a polypeptide chain will have a free amino group, and the opposite end will have a free carboxy group. During elongation, the ribosome reads the mRNA templates from the 5′ end to the 3′ end, adding amino acids sequentially to the C-terminus of the growing chain. Multiple ribosomes can translate the same mRNA simultaneously, forming polyribosomes for efficient translation.
Translation Termination
This occurs when a stop codon (UGA, UAG, or UAA) enters the A site of the ribosome. The process is facilitated by eukaryotic release factors (eRFs). Instead of a tRNA, the eRF1 recognizes all stop codons, assisted by eRF3, a GTPase that helps release the polypeptide chain. This leads to dissociation of ribosomal subunits, and they are recycled. Like other processes, cells maintain quality control mechanisms through various mechanisms, like nonsense-mediated mRNA decay (NMD), which targets premature stop codons by sensing the distance between stop codons and exon junction complexes, while non-stop decay targets and degrades mRNAs that lack stop codons by recruiting the degradation machinery via the ski7 complex.
Stop codon read-through is widespread in monocots and dicots, allowing stop codons to be read as 20 standard amino acids. This occurs in transcripts with simpler structures and is not evolutionarily conserved. This reveals the remarkable plasticity and regulatory potential of stop codon usage in plant gene expression.
For more information read: Readthrough events in plants reveal plasticity of stop codons
Amino Acid Properties
Amino acids are the building blocks of proteins (Figure 20). Except for the amino acid proline, they all consist of a central carbon atom covalently bonded to an amino group, a hydrogen atom, a carboxyl group, and a variable R group. In the physiological range, both the carboxylic acid and amino groups are completely ionized, allowing amino acids to act as either an acid or a base. Thus, amino acids do not assume a neutral form in an aqueous environment.
The chemical and physical properties of the side chains provide functionally important properties to amino acids. Some of the important properties of side chains include acidic, basic, or neutral, hydrophobic vs. hydrophilic, and the size of side chains. They are categorized according to their side groups as nonpolar, polar, positively or negatively charged, or aromatic. All these properties and others affect how each amino acid contributes to the structure and function of a mature protein.
Protein Structure and Function
Proteins assume complex 3-dimensional structures essential for their functions as enzymes, regulatory factors, or structural proteins. Complex physical-chemical interactions among amino acid side groups determine protein structure.
Several levels of structure are considered. The primary structure of a protein is the amino acid sequence of its polypeptide chains. The secondary structure is derived through the interactions between neighboring amino acids to form local structural elements such as beta sheets or alpha helices. The tertiary structure refers to the three-dimensional structure of an entire polypeptide and is determined by the diverse properties of amino acid side groups. For example, in an aqueous environment, hydrophobic amino acids will often interact at the core of a protein structure, while hydrophilic groups may be exposed to the protein surface.
Many proteins are multimeric, composed of several polypeptide chains called “subunits”, which associate non-covalently or in some cases via disulfide bonds. Such higher-order spatial associations among polypeptides result from interactions among amino acids and result in a quaternary structure. Some multimeric proteins consist of multiple copies of the same polypeptide. Alcohol dehydrogenase, important for flood tolerance, is a homodimer containing two subunits of the same polypeptide encoded at the same gene locus (Schwartz, 1966). Other proteins contain different peptides. ADP-glucose pyrophosphorylase, the rate-limiting enzyme in maize kernel starch synthesis, is a heterodimer containing two different polypeptides encoded by two distinct genes, shrunken2 and brittle2 (Hannah, 2005).
For more information read: Starch synthesis in the maize endosperm
For more information read: The genetic control of alcohol dehydrogenase in maize: gene duplication and repression
Chapter Summary
DNA is composed of nucleotides. Phosphate bonds connect nucleotides to form a strand. The bases on one strand form hydrogen bonds with the bases on the other to form a double-stranded molecule. The final feature of the molecular structure is that DNA assumes a helical conformation. To fit inside the nucleus, DNA assumes a condensed structure. Therefore, chromosomal DNA is coiled around a histone protein core to form chromatin. The tight packaging of DNA in chromatin must be modified to allow DNA replication and transcription. In the process of replication, the two DNA strands separate and act as templates for the synthesis of complementary daughter strands. The genetic information of DNA is transferred through transcription to an intermediate molecule called RNA. The starting and stopping of transcription are controlled by specific DNA sequence elements that are located within the DNA sequence and referred to as promoter and terminator sequences. The coding region of a gene is composed of a sequence of nucleotides that are transcribed into RNA. These sequences include exons and introns. Exons are the sequences that code for proteins. The coding region of a gene contains exons and introns. Also, pre-mRNA contains both introns and exons. The introns in pre-mRNA are removed through a process called intron splicing. The mRNA is processed by 5′ capping and addition of a poly(A) tail. Mature RNA is then translated into amino acids used to build proteins.
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