G

nucleotide excision repair, and postreplication repair (77,78). Several steps in the process of DNA repair are shared by these multiple pathways, including (1) recognition of sites of damage, (2) removal of damaged nucleotides, and (3) restoration of the normal DNA sequence. Each of these steps in DNA repair are accomplished by specific proteins and enzymes. Surveillance of the cellular DNA is a continual process involving specific aspects of the transcription and replication machinery. However, in each case where the restoration of the normal DNA sequence is accomplished through the replacement of damaged nucleotides, the undamaged DNA strand serves as a template in the repair process. This ensures the faithful reproduction of the primary structure of the DNA at the damaged site.

8. CHEMICAL NATURE OF RNA

Like DNA, RNA is composed of repeating purine and pyrim-idine nucleotide subunits. However, several distinctions can be made with respect to the chemical nature of RNA and DNA. Unlike the 2'-deoxyribose sugar moiety of DNA, the sugar moiety in RNA is ribose. Like DNA, RNA usually contains adenine, guanine, and cytosine, but does not contain thymidine. In place of thymidine, RNA contains uracil. The concentration of purines and pyrimidine bases do not necessarily equal one another in RNA because of the single-stranded nature of the molecule. The monomeric units of RNA are linked together by 3',5'-phosphodi-ester bonds analogous to those in DNA (Fig. 3). RNAs have molecular weights between 1 x 104 Daltons for transfer RNA (tRNA) and 1 x 107 Daltons for ribosomal RNA (rRNA).

9. STRUCTURE AND FUNCTION OF RNA

RNA exists as a long, regular, unbranched polynucleotide strand. The informational content of the RNA molecule is contained in its primary structure or nucleotide sequence. In spite of the fact that RNA exists primarily as a single-stranded molecule, significant higher-order structures are often formed in individual RNA molecules. In some cases, this higher-order structure is related to the actual function of the molecule. Three major classes of RNA are found in eukaryotic organisms: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Each class differs from the others in the size, function, and general stability of the RNA molecules (79). Minor classes of RNA include heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), and small cytoplasmic RNA (scRNA).

9.1. STRUCTURE AND FUNCTION OF MESSENGER RNA The ability of DNA to serve directly as a template for the synthesis of protein is precluded by the observations that protein synthesis takes place in the cytoplasm, whereas almost all of the cellular DNA resides in the nucleus. Thus, genetic information contained in the DNA must be transferred to an intermediate molecule that is translocated into the cell cytoplasm, where it directs the ordering of amino acids in protein synthesis. RNA fulfills this role as the intermediate molecule for the transport and translation of genetic information (11). Messenger RNA molecules represent transcripts of structural genes that encode all of the information necessary for the synthesis of a single-type polypeptide of protein. Thus, mRNAs serve two important functions with respect to protein synthesis: (1) mRNAs deliver genetic information to the cytoplasm where protein synthesis takes place and (2) mRNAs serve as a template (or blueprint) for translation by ribosomes during protein synthesis.

Mammalian cells (among others) express "interrupted" genes; that is, genes with coding sequences are not contiguous (continuous) in the DNA, and that require a posttranscriptional modification prior to translation of protein products (Fig. 4). The majority of structural genes in the higher eukaryotic organisms are interrupted. The average gene contains 7-10 exons, spread over 10-20 kb of DNA. For instance, the p53 tumor suppressor gene is composed of 11 exons, occupies approx 16 kb in the genomic DNA, and produces a 2-kb mRNA (80-82). However, other genes are much larger. For example, the Rb1 tumor suppressor gene occupies 200 kb in the genomic DNA, contains 27 exons, and gives rise to a 4.7-kb mRNA (83-85). The primary RNA transcript exhibits the same overall structure and organization as the structural gene and is often referred to as the pre-mRNA. Removal of intronic sequences yields a mature mRNA that is considerably smaller with an average size of 1-3 kb. The process of removing the intronic sequences is called RNA splicing (86-88).

The primary products of RNA transcription in the nucleus compose a special class of RNAs that are characterized by their large size and heterogeneity (79). These RNA molecules are referred to as heterogeneous nuclear RNAs (hnRNAs). Heterogeneous nuclear RNAs contain both intronic and exonic sequences encoded in the template DNA of structural genes. These hnRNAs are processed in the nucleus (87,89,90) to give mature mRNAs that are transported into the cytoplasm, where they participate in protein synthesis. Nuclear processing of RNA involves (1) chemical modification reactions (addition of the 5' CAP), (2) splicing reactions (removal of intronic sequences), and (3) polyadenylation [addition of the 3' poly(A) tail]. Additional processing of some specific mRNAs occurs in the cell cytoplasm, including RNA editing reactions (91-93). It has been suggested that some snRNAs function in the processing of hnRNAs (94). Mature mRNAs are transported into the cytoplasm of the cell, where they participate in the translational processes of protein synthesis.

In RNA splicing, intronic sequences are specifically removed from the primary RNA transcript and the remaining exonic sequences are rejoined into one molecule. There is no extensive homology or complementarity between the two ends of an intron precluding the general possibility that intronic sequences form extensive secondary structures (such as a hairpin loop) as a preliminary step in the splicing reaction. The splice junctions represent short, well-conserved consensus sequences. The generic intron contains a GT sequence at the 5' boundary and an AG sequence at the 3' boundary (Fig. 5). The 5' and 3' splice junctions are often referred to as the splice donor and splice acceptor sites, respectively. Splice sites are generic in that they do not

Mature RNA Transcript Fig. 5. Fundamental aspects of RNA splicing.

exhibit specificity for individual RNA precursors, and individual precursors do not convey specific information that is required for splicing. In principle, any splice donor site can react with any splice acceptor site. However, under normal conditions, these reactions are restricted to the donor and acceptor sites of the same intron. Analysis of molecular intermediates formed during the splicing of large precursor RNAs suggests that the introns are removed in a definitive pattern or through a preferred pathway that dictates the general order of intron removal. This suggestion might imply a mechanism in which the conformation of precursor RNA molecules limits the accessibility of splice junctions, such that as specific introns are removed, the conformation of the molecule changes and new splice sites become available. However, several other plausible mechanims exists that could account for the observed patterns of splicing in RNAs that have been examined in detail (86,89,90). The RNA sequences that are required for successful splicing include (1) the 5' splice donor and 3' splice acceptor consensus sequences and (2) a consensus sequence known as the branch site. The branch site is located approx 20-40 bases upstream of the 3'-terminus of the intronic sequence and conforms to the following consensus sequence: Py-N-Py-Py-Pu-A-Py. The role of the branch site is to identify the nearest splice acceptor site for connection to the splice donor site. The first step in splicing involves a cleavage of the RNA molecule at the 5' end of the intron. The resulting intron-exon molecule forms a structure known as a lariat, through the formation of a 5'-2' bond between the G residue located at the 5' end of the intron and the adenine residue of the branch site. The second step involves cleavage of the RNA molecule at the 3' splice site This cleavage releases the intron in lariat form, which is subsequently degraded.

The 5'-termini of all eukaryotic mRNAs are modified post-tracriptionally, through enzymatic reactions occuring in the nucleus and in the cytoplasm of the cell. Initially, a guanine residue is added to the 5'-termini of primary mRNA transcripts through the action of an enzyme called guanylyl transferase. This reaction occurs in the nucleus soon after the intitiation of transcription. This guanine residue is linked to the first coded nucleoside triphosphate through a 5'-5' triphosphate linkage, rather than a 3'-5' phosphodiester bond. Thus, this guanine residue occurs in the structure of the mRNA in reverse orientation from all the other nucleotides. The modified 5'-terminus is referred to as a "CAP" and is the site of additional modification reactions involving the addition of methyl groups. These additional modifications are catalyzed by enzymes located in the cytoplasm of the cell. The first methylation occurs in all mRNAs and consists of the addition of a methyl group to the 7-position of the terminal guanine through the action of guanine-7-methyl-transferase. Additional methylation reactions can occur involving the additional bases at the 5'-terminus of the mRNA transcript, and less frequently, internal methylation of bases within an mRNA molecule takes place.

The poly(A) tail possessed by most eukaryotic mRNAs is not encoded in the DNA; rather, it is added to the RNA in the nucleus after transcription of the structural gene is complete. The addition of the poly(A) tail is catalyzed by the enzyme poly(A) polymerase, which adds approx 200 adenosine residues to the free 3'-OH terminus of the primary RNA transcript. The precise function of the poly(A) tail is unknown, but has been speculated to be involved in mRNA stability or in control of mRNA utilization (95). Although the removal of the poly(A) tail does precede degradation of certain mRNAs, a systematic correlation between mRNA stability or survival, and the length or presence of the poly(A) tail has not been established. Removal of the poly(A) tail can inhibit the initiation of translation in vitro, suggesting a potential role for this structure in the control of mRNA translation. However, it is not clear whether this effect is related to a direct influence of poly(A) structures on the initiation reaction or the result of some indirect cause.

Some other forms of posttranscriptional RNA processing occur with respect to a small subset of eukaryotic mRNAs. The process of RNA editing involves a posttranscriptional alteration of the informational content of a specific mRNA (91). The editing of RNA is revealed when the linear sequence of the mRNA molecule differs from the coding sequence carried in the DNA. In mammalian cells, there are examples where substitution of a single base occurs in the mRNA, resulting in an alteration of an amino acid in the final protein product. Because no known template source mediates the RNA editing reaction, the most likely mechanism for mRNA editing would involve a specific enzyme that can recognize the sequence or secondary structure of the specific target mRNA and catalyze the specific base substitution. However, there are examples of RNA editing in some lower eukaryotes that utilize "guide RNA," which directs the RNA editing reaction (96). The final result of RNA editing is the generation protein products representing more than one polypeptide sequence from a single coding gene. The different protein products might possess different biological activities, suggesting that RNA editing might represent a mechanism for controlling the functional expression of genes through a post-transcriptional process that does not impact the normal mechanisms for controlling levels of gene expression.

9.2. STRUCTURE AND FUNCTION OF TRANSFER RNA Transfer RNAs are small molecules consisting of approx 75-80 nucleotides. Like mRNAs, tRNA is generated through nuclear processing of precursor RNA transcripts. The structure of the tRNA molecule reflects its function as an adapter between the mRNA and amino acids during protein synthesis. Specific tRNAs correspond to each of the amino acids utilized in protein synthesis in any particular cell type. Although the specific tRNAs differ from each other with respect to their actual nucleotide sequence, tRNAs as a class of RNA molecules share several common structural features. Each tRNA contains information in the primary structure or nucleotide sequence that dictates the higher-order structure of the molecule. The secondary structure of the tRNA resembles a cloverleaf (97). The folding of the cloverleaf structure is maintained through intrastrand sequence complementarity and base-pairing interactions between nucleotides. In addition, each tRNA contains an ACC sequence at the 3'-terminus and an anti-codon loop (97). Amino acids are attached to their specific tRNA through an ester bond to the 3'-hydroxyl group of the terminal adenine of the ACC sequence. The anticodon loop recognizes the triplet codon of the template mRNA during the process of translation. With the exception of the codons encoding methionine and tryptophan, there are at least two possible codons for each amino acid (Table 1). Nonetheless, each amino acid has only one corresponding tRNA. Thus, the hydrogen-bonding between nucleotides of the codon and anticodon often involve "wobble" pairing (98). This form of base-pairing allows mismatches in the third base of a codon triplet. In the overall structure-function relationship in tRNA, the nucleotide sequence of the anticodon loop dictates which amino acid will be attached to the ACC sequence of the tRNA.

Transfer RNAs serve as adapters between the mRNA template and the amino acids of growing polypeptide chains during the process of protein translation (97); that is, the tRNA serves to ferry the appropriate amino acid into the active site of the ribo-some, where it becomes incorporated into the growing polypep-tide being synthesized. Amino acids are coupled to their specific tRNA through the action of enzymes called aminoacyl tRNA synthetases. The specificity of the "charging" reaction is critical to the integrity of the translation process because the incorporation of amino acids at the level of the ribosome depends wholly on the sequence of the anticodon portion of the tRNA molecule. The charged tRNA the mRNA through the transient hybridization of the codon and anti-codon RNA sequences in the ribosome complex as it moves along the mRNA. The entry of the charged tRNA into the active site of the ribosome brings its associated amino acid into juxtaposition with the nascent polypeptide, facilitating the formation of a peptide bond. In this manner, the tRNA provides a link between the genetic information contained in the mRNA and the linear sequence of amino acids represented in the resulting polypeptide product.

9.3. STRUCTURE AND FUNCTION OF RIBOSOMAL RNA The ribosome is a nucleoprotein that serves as the primary component of a cell's protein synthesis machinery. The ribosome is a complex structure consisting of two subunit particle types (60S and 40S). The overall composition of the fully assembled ribosome includes at least 4 distinct rRNA molecules and nearly 100 specific protein subunits. The major rRNAs in mammalian cells were named for their molecular size as determined by their sedimentation rates. Three of these rRNAs (5S,

Table 2

Secondary and Tertiary Structural Motifs in RNA

Structure

Description

Functions

Hairpin loops

Internal loops

Bulges

Nucleotide triples

Pseudoknots

Single-stranded loop that bridges one end of a double-stranded stem

Interruptions in double-stranded RNA caused by the presence of nucleotides on both strands that cannot participate in Watson-Crick base-pairing

Double-stranded RNA molecules with unpaired nucleotides on only one strand

Triple helical structures that form through hydrogen bonding between nucleotides of a single-stranded RNA molecule and nucleotides within a double-stranded RNA molecule; hydrogen bonds can involve nucleotide bases, sugars, or phosphate groups

Results from base-pairing between nucleotide sequences within an RNA loop structure and a complementary nucleotide sequence outside the RNA loop

Component of more complex RNA structures; May serve as a nucleation site for RNA folding, or recognition sites for protein-RNA interaction

Protein-binding sites and ribozyme cleavage sites

Contribute to the formation of more complex RNA structures; recognition sites for protein-RNA interactions.

Orient regions of secondary structure in large RNA molecules and stabilize three-dimensional RNA structures.

RNA self-splicing, autoregulation of translational processes, and ribosome frameshifting

5.8S, and 28S) are components of 60S ribosomal particle. The smaller 40S ribosomal particle contains a single 18S rRNA. The 5.8S, 18S, and 28S rRNAs are the products of the processing of a single 45S precursor RNA molecule. The 5S rRNA is independently transcribed and processed. The rRNAs assemble with ribo-somal protein subunits in the nucleus. The precise role of rRNA in the function of the ribosome is not completely understood (99). However, it is recognized that interactions between the rRNAs of the ribosomal subunits might be important in the overall structure of the functioning ribosomal particle. In addition, rRNA sequences can interact with ribosome-binding sequences of mRNA during the initiation of translation. Likewise, it is likely that rRNAs bind to invariant tRNA sequences when these molecules enter the active site of the ribosome (99).

9.4. SPECIAL RNA STRUCTURES Higher-order RNA structures exhibit hydrogen-bonding between A:U and G:C basepairs. Several specific higher-order RNA structures have been recognized (Table 2) and characterized in detail (100). Hairpin loops consist of a double-stranded stem and a single-stranded loop that bridges one end of the stem. These structures are essential components of more complex RNA structures and probably serve as nucleation sites for RNA folding. Loops can also function as recognition sites for protein-RNA interactions. Internal loops represent interruptions in double-stranded RNA caused by the presence of nucleotides on both strands that cannot participate in Watson-Crick base-pairing. Several important functions are associated with internal loops, including protein-binding sites and ribozyme cleavage sites. In many cases, internal loops have been shown to represent highly ordered structures maintained by the formation of non-Watson-Crick basepairs. Bulges are structural motifs contained within double-stranded RNA molecules with unpaired nucleotides on only one strand. RNA bulges contribute to the formation of more complex, higher-order RNA structures and can also serve as recognition sites for protein-RNA interaction. Nucleotide triples occur when single-stranded RNA sequences form hydrogen bonds with nucleotides that are already base-paired. These interactions serve to stabilize three-dimensional RNA structures and to orient regions of RNA secondary structure in large RNA molecules. Pseudoknots are tertiary structural elements that result from base-pairing between nucleotide sequences contained within a loop structure and sequences outside the loop structure. These are important in RNA self-splicing, translational autoregulation, and ribosomal frameshifting.

10. DNA DAMAGE, MUTAGENESIS, AND THE CONSEQUENCES OF MUTATION

DNA damage can result from spontaneous alteration of the DNA molecule or from the interaction of numerous chemical and physical agents with the structural DNA molecule (101). Spontaneous lesions can occur during normal cellular processes, such as DNA replication, DNA repair, or gene rearrangement (78), or through chemical alteration of the DNA molecule itself as a result of hydrolysis, oxidation, or methyla-tion (102,103). In most cases, DNA lesions create nucleotide mismatches that lead to point mutations. Nucleotide mismatches can result from the formation of apurinic or apyrimidinic sites following depurination or depyrimidination reactions (103), nucleotide conversions involving deamination reactions (78), or, in rare instances, from the presence of a tautomeric form of an individual nucleotide in replicating DNA. Deamination reactions can result in the conversion of cytosine to uracil, adenine to hypoxanthine, and guanine to xanthine (78). However, the most common nucleotide deamination reaction involves methylated cytosines, which can replace cytosine in the linear sequence of a DNA molecule in the form of 5-methylcytosine. The 5-methylcytosine residues are always located next to guanine residues on the same chain, a motif referred to as a CpG island. The deamination of 5'-methylcytosine results in the formation of thymine. This particular deamination reaction accounts for a large percentage of spontaneous mutations in human disease (104-106). Interaction of DNA with physical agents, such as ionizing radiation, can lead to single- or double-strand breaks as a result of scission of phosphodiester bonds on one or both polynucleotide strands of the DNA molecule (78). Ultraviolet (UV) light can produce different forms of photoproducts, including pyrimidine dimers between adjacent pyrimidine bases on the same DNA strand. Other minor forms of DNA damage caused by UV light include strand breaks and crosslinks (78). Nucleotide base modifications can result from exposure of the DNA to various chemical agents, including N-nitroso compounds and polycyclic aromatic hydrocarbons (78). DNA damage can also be caused by chemicals that intercalate the DNA molecule and/or crosslink the DNA strands (78). Bifunctional alkylating agents can cause both intrastrand and interstrand crosslinks in the DNA molecule.

The various forms of spontaneous and induced DNA damage can give rise to a plethora of different types of molecular mutation (107). These various types of mutation include both gross alteration of chromosomes and more subtle alterations to specific gene sequences in otherwise normal chromosomes. Gross chromosomal aberrations include (1) large deletions, (2) additions (reflecting amplification of DNA sequences), and (3) translocations (reciprocal and nonreciprocal). All of these forms of chromosomal abnormality can be distinguished through standard karyotype analyses of G-banded chromosomes (Fig. 6). The major consequence of chromosomal deletion is the loss of specific genes that are located in the deleted chromosomal region, resulting in changes in the copy number of the affected genes. The deletion of certain classes of genes such as tumor suppressor genes or genes encoding the proteins involved in DNA repair can predispose cells to neoplastic transformation (108,109). Likewise, amplification of chromosomal regions results in an increase in gene copy numbers, which can lead to the same type of circumstance if the affected region contains genes for dominant proto-oncogenes or other positive mediators of cell cycle progression and proliferation (108-110). The direct result of chromosomal translocation is the movement of some segment of DNA from its natural location into a new location within the genome, which can result in altered expression of the genes that are contained within the translocated region. If the chromosomal breakpoints utilized in a translocation are located within structural genes, then new hybrid genes can be generated.

The most common forms of mutation involve single-nucleotide alterations, small deletions, or small insertions into specific gene sequences. These microscopic alterations very often can only be detected through DNA sequencing. Single-nucleotide alterations that involve a change in the normal coding sequence of the gene are referred to as point mutations. The consequence of most point mutations is an alteration in the amino acid sequence of the encoded protein. However, some point mutations are "silent" and do not affect the structure of the gene product (Table 3). Silent mutations are possible because most amino acids can be encoded by more than one triplet codon (Table 1). Point mutations fall into two classes:

Fig. 6. Forms of gross chromosomal aberrations. Three examples of chromosomal aberrations are demonstrated using a standard G-band ideogram of human chromosome 11. The asterisks denote the chromosomal band that has expanded as a result of DNA amplification. The stippled area highlights a region from another chromosome that has been translocated to the long arm of the starting chromosome, displacing the normal qter region without altering the overall size of the chromosome (note the differences in G-banding in this region).

Fig. 6. Forms of gross chromosomal aberrations. Three examples of chromosomal aberrations are demonstrated using a standard G-band ideogram of human chromosome 11. The asterisks denote the chromosomal band that has expanded as a result of DNA amplification. The stippled area highlights a region from another chromosome that has been translocated to the long arm of the starting chromosome, displacing the normal qter region without altering the overall size of the chromosome (note the differences in G-banding in this region).

missense mutations and nonsense mutations. Missense mutations involve nucleotide base substitutions that alter the translation of the affected codon triplet. In contrast, nonsense mutations involve nucleotide base substitutions that modify a triplet codon that normally encodes for an amino acid into a

Table 3

Forms and Consequences of Molecular Mutation

Normal coding sequence and amino acid translation

Missense point mutation resulting in amino acid change

Missense point mutation without amino acid change (silent mutation)

Frameshift mutation resulting from a single base insertion

Frameshift mutation resulting from a single base deletion

Nonsense mutation resulting in a premature stop codon

Asn AAU

Asn AAU

Asn AAU

My First Baby

My First Baby

Are You Prepared For Your First Baby? Endlessly Searching For Advice and Tips On What To Expect? Then You've Landed At The Right Place With All The Answers! Are you expecting? Is the time getting closer to giving birth to your first baby? So many mothers to be are completely unprepared for motherhood and the arrival of a little one, but stress not, we have all the answers you need!

Get My Free Ebook


Post a comment