Cell and Molec Bio Chapter 6, 7, 8

Ribozymes

RNA molecules that function as enzymes. The ribosome in protein translation is a ribozyme. RNA molecules that possess catalytic activity are called ribozymes.

Messelson-Stahl Experiment

semiconservative replication

polyribosomes aka polysomes

Strings of ribosomes that work together to translate a RNA message. Polysomes operate in both bacteria and eukaryotes, but bacteria can speed up the rate of protein synthesis even further. Because bacterial mRNA does not need to be processed and is also physically accessible to ribosomes while it is being made, ribosomes will typically attach to the free end of a bacterial mRNA molecule and start translating it even before the transcription of that RNA is complete; these ribosomes follow closely behind the RNA polymerase as it moves along DNA.

single stranded DNA binding proteins

Single- strand DNA-binding proteins cling to the single-stranded DNA exposed by the helicase, transiently preventing the strands from re-forming base pairs and keeping them in an elongated form so that they can serve as efficient templates.

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

thymine dimers

The ultraviolet radiation in sunlight is also damaging to DNA; it promotes covalent linkage between two adjacent pyrimidine bases, forming, for example, the thymine dimer shown in Figure 6-24. It is the failure to repair thymine dimers that spells trouble for individuals with the disease xeroderma pigmentosum.

3'end of tRNA

This is the site where the amino acid that matches the codon is covalently attached to the tRNA.

tRNA binding sites

the tRNA, sites are designated the a, p, and e sites (short for aminoacyl- tRNA, peptidyl-tRNA, and exit, respectively).

DNA repair basic pathways

1. The damaged DNA is recognized and removed by one of a variety of mechanisms. These involve nucleases, which cleave the cova- lent bonds that join the damaged nucleotides to the rest of the DNA strand, leaving a small gap on one strand of the DNA double helix in the region. 2. A repair DNA polymerase binds to the 3ʹ-hydroxyl end of the cut DNA strand. It then lls in the gap by making a complementary copy of the information stored in the undamaged strand. Although different from the DNA polymerase that replicates DNA, repair DNA polymerases synthesize DNA strands in the same way. For example, they elongate chains in the 5ʹ-to-3ʹ direction and have the same type of proofreading activity to ensure that the template strand is copied accurately. In many cells, this is the same enzyme that lls in the gap left after the RNA primers are removed during the normal DNA replication process (see Figure 6-17). 3. When the repair DNA polymerase has lled in the gap, a break remains in the sugar-phosphate backbone of the repaired strand. This nick in the helix is sealed by DNA ligase, the same enzyme that joins the Okazaki fragments during replication of the lagging DNA strand. Steps 2 and 3 are nearly the same for most types of DNA damage, including the rare errors that arise during DNA replication. However, step 1 uses a series of different enzymes, each specialized for removing different types of DNA damage. Humans produce hundreds of different proteins that function in DNA repair.

guanine triphosphate (GTP)

energy-providing molecule that binds to eIF-2 and is needed for translation

chaperone proteins

help new proteins fold into their normal shape.

RNA

Although their chemical differences are small, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. This difference has important functional consequences. Because an RNA chain is single- stranded, it can fold up into a variety of shapes, just as a polypeptide chain folds up to form the final shape of a protein (Figure 7-5); double- stranded DNA cannot fold in this fashion. As we discuss later, the ability to fold into a complex three-dimensional shape allows RNA to carry out various functions in cells, in addition to conveying information between DNA and protein. Whereas DNA functions solely as an information store, some RNAs have structural, regulatory, or catalytic roles.

Deamination

Another common reaction is the spontaneous loss of an amino group (deamination) from a cytosine in DNA to produce the base uracil (Figure 6-23B). Some chemically reactive by-products of cell metabolism also occasionally react with the bases in DNA, altering them in such a way that their base-pairing properties are changed.

DNA topoisomerase

As the helicase pries open the DNA within the replication fork, the DNA on the other side of the fork gets wound more tightly. This excess twisting in front of the replication fork creates tension in the DNA that—if allowed to build—makes unwinding the double helix increasingly dif - cult and impedes the forward movement of the replication machinery. Cells use proteins called DNA topoisomerases to relieve this tension. These enzymes produce transient nicks in the DNA backbone, which temporarily release the tension; they then reseal the nick before falling off the DNA (Figure 6-20B

mRNA molecules Are eventually Degraded in the Cytosol

Because a single mRNA molecule can be translated into protein many times (see Figure 7-2), the length of time that a mature mRNA mole- cule persists in the cell affects the amount of protein it produces. Each mRNA molecule is eventually degraded into nucleotides by ribonucleases (RNAses) present in the cytosol, but the lifetimes of mRNA molecules dif- fer considerably—depending on the nucleotide sequence of the mRNA and the type of cell. In bacteria, most mRNAs are degraded rapidly, hav- ing a typical lifetime of about 3 minutes. The mRNAs in eukaryotic cells usually persist longer: some, such as those encoding β-globin, have life- times of more than 10 hours, whereas others have lifetimes of less than 30 minutes. These different lifetimes are in part controlled by nucleotide sequences in the mRNA itself, most often in the portion of RNA called the 3′ untrans- lated region, which lies between the 3′ end of the coding sequence and the poly-A tail. The different lifetimes of mRNAs help the cell control the amount of each protein that it synthesizes. In general, proteins made in large amounts, such as β-globin, are translated from mRNAs that have long lifetimes, whereas proteins made in smaller amounts, or whose lev- els must change rapidly in response to signals, are typically synthesized from short-lived mRNAs.

Nucleoside Triphosphate (NTP)

Building unit for RNA transcription (ribonucleotides) and DNA replication (deoxyribonucleotides);, a ribose nucleotide with 2 additional phosphates, which are chopped off during RNA- synthesis process.

CHAPTER 7

CHAPTER 7

transcription factors (eukaryotes).

Collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription (eukaryotes). Principal among these are the general transcrip- tion factors, which must assemble at each promoter, along with the polymerase, before the polymerase can begin transcription.

DNA repair

Collective term for the enzymatic processes that correct deleterious changes affecting the continuity or sequence of a DNA molecule.--The thousands of random chemical changes that occur every day in the DNA of a human cell—through thermal collisions or exposure to reac- tive metabolic by-products, DNA-damaging chemicals, or radiation—are repaired by a variety of mechanisms, each catalyzed by a different set of enzymes.

replication origin

The process of DNA synthesis is begun by initiator proteins that bind to speci c DNA sequences called replication origins. Here, the initiator proteins pry the two DNA strands apart, breaking the hydrogen bonds between the bases (Figure 6-4). Although the hydrogen bonds collec- tively make the DNA helix very stable, individually each hydrogen bond is weak (as discussed in Chapter 2). Separating a short length of DNA a few base pairs at a time therefore does not require a large energy input, and the initiator proteins can readily unzip the double helix at normal temperatures.----The human genome, which is very much larger than a bacterial genome(and has a single replication origin), has approximately 10,000 such origins—an average of 220 origins per chromosome. Beginning DNA replication at many places at once greatly shortens the time a cell needs to copy its entire genome

DNA polymerase

Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule. The movement of a replication fork is driven by the action of the replica- tion machine, at the heart of which is an enzyme called DNA polymerase. This enzyme catalyzes the addition of nucleotides to the 3ʹ end of a grow- ing DNA strand, using one of the original, parental DNA strands as a template. Base pairing between an incoming nucleotide and the template strand determines which of the four nucleotides (A, G, T, or C) will be selected. The nal product is a new strand of DNA that is complementary in nucleotide sequence to the template (Figure 6-10). The polymerization reaction involves the formation of a phosphodiester bond between the 3ʹ end of the growing DNA chain and the 5ʹ-phosphate group of the incoming nucleotide, which enters the reaction as a deoxy- ribonucleoside triphosphate. The energy for polymerization is provided by the incoming deoxyribonucleoside triphosphate itself: hydrolysis of one of its high-energy phosphate bonds fuels the reaction that links the nucleotide monomer to the chain, releasing pyrophosphate (Figure 6-11). Pyrophosphate is further hydrolyzed to inorganic phosphate (Pi), which makes the polymerization reaction effectively irreversible

codon

In 1961, it was discovered that the sequence of nucleotides in an mRNA molecule is read consecutively in groups of three. And because RNA is made of 4 different nucleotides, there are 4 × 4 × 4 = 64 possible combi- nations of three nucleotides: AAA, AUA, AUG, and so on. However, only 20 different amino acids are commonly found in proteins. Either some nucleotide triplets are never used, or the code is redundant, with some amino acids being speci ed by more than one triplet. The second pos- sibility turned out to be correct, as shown by the completely deciphered genetic code shown in Figure 7-25. Each group of three consecutive nucleotides in RNA is called a codon, and each codon speci es one amino acid. The strategy by which this code was cracked is described in How We know, pp. 240-241.

sigma factor

In bacteria, it is a subunit of RNA polymerase, the sigma (σ) factor (see Figure 7-9), that is primarily responsible for recognizing the promoter sequence on the DNA. But how can this factor "see" the promoter, given that the base-pairs in question are situated in the interior of the DNA double helix? It turns out that each base presents unique features to the outside of the double helix, allowing the sigma factor to nd the promoter sequence without having to separate the entwined DNA strands. ---see figure---Bacterial rNa polymerase (light blue) contains a subunit called sigma factor (yellow) that recognizes the promoter of a gene (green). Once transcription has begun, sigma factor is released, and the polymerase moves forward and continues synthesizing the rNa. chain elongation continues until the polymerase encounters a sequence in the gene called the terminator (red ). there the enzyme halts and releases both the DNa template and the newly made rNa transcript. the polymerase then reassociates with a free sigma factor and searches for another promoter to begin the process again.

Introns

In bacteria, most proteins are encoded by an uninter- rupted stretch of DNA sequence that is transcribed into an mRNA that, without any further processing, can be translated into protein.

translation initiation factors (eukaryotes)

In eukaryotes, an initiator tRNA, charged with methionine, is rst loaded into the P site of the small ribosomal subunit, along with additional pro- teins called translation initiation factors (Figure 7-36). The initiator tRNA is distinct from the tRNA that normally carries methionine. Of all the tRNAs in the cell, only a charged initiator tRNA molecule is capable of binding tightly to the P site in the absence of the large ribosomal subunit. Next, the small ribosomal subunit loaded with the initiator tRNA binds to the 5′ end of an mRNA molecule, which is marked by the 5′ cap that is present on all eukaryotic mRNAs (see Figure 7-16). The small ribosomal subunit then moves forward (5′ to 3′) along the mRNA searching for the rst AUG. When this AUG is encountered and recognized by the initiator tRNA, several initiation factors dissociate from the small ribosomal sub- unit to make way for the large ribosomal subunit to bind and complete ribosomal assembly. Because the initiator tRNA is bound to the P site, protein synthesis is ready to begin with the addition of the next charged tRNA to the A site (see Figure 7-34).

gene expression

In the broadest sense, the term gene expression refers to the process by which the information encoded in a DNA sequence is translated into a product that has some effect on a cell or organism. In cases where the nal product of the gene is a protein, gene expression includes both transcription and translation. When an RNA molecule is the gene's nal product, however, gene expression does not require translation.

RNA polymerase

Like the DNA polymerase that carries out DNA replication (discussed in Chapter 6), RNA polymerases catalyze the formation of the phos- phodiester bonds that link the nucleotides together and form the sugar-phosphate backbone of the RNA chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead to expose a new region of the template strand for comple- mentary base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5′-to-3′ direction (Figure 7-7). The incom- ing ribonucleoside triphosphates (ATP, CTP, UTP, and GTP) provide the energy needed to drive the reaction forward (see Figure 6-11). The almost immediate release of the RNA strand from the DNA as it is syn- thesized means that many RNA copies can be made from the same gene in a relatively short time; the synthesis of the next RNA is usually started before the rst RNA has been completed (Figure 7-8). A medium-sized gene—say, 1500 nucleotide pairs—requires approximately 50 seconds for a molecule of RNA polymerase to transcribe it (Movie 7.2). At any given time, there could be dozens of polymerases speeding along this single stretch of DNA, hard on one another's heels, allowing more than 1000 transcripts to be synthesized in an hour. For most genes, however, the amount of transcription is much less than this.

Transfer RNA

The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for exam- ple, bind directly to the amino acid. Rather, the translation of mRNA into protein depends on adaptor molecules that can recognize and bind to a codon at one site on their surface and to an amino acid at another site. These adaptors consist of a set of small RNA molecules known as trans- fer RNAs (tRNAs), each about 80 nucleotides in length.

DNA helicase

The helicase sits at the very front of the replication machine where it uses the energy of ATP hydrolysis to propel itself forward, prying apart the double helix as it speeds along the DNA

general transcription factors

The initial nding that, unlike bacterial RNA polymerase, puri ed eukary- otic RNA polymerase II could not initiate transcription on its own in a test tube led to the discovery and puri cation of the general transcrip- tion factors. These accessory proteins assemble on the promoter, where they position the RNA polymerase and pull apart the DNA double helix to expose the template strand, allowing the polymerase to begin tran- scription. Thus the general transcription factors have a similar role in eukaryotic transcription as sigma factor has in bacterial transcription. The assembly process typically begins with the binding of the general transcription factor TFIID to a short segment of DNA double helix composed primarily of T and A nucleotides; because of its composition, this part of the promoter is known as the TATA box. Upon binding to DNA, TFIID causes a dramatic local distortion in the DNA double helix (Figure 7-13), which helps to serve as a landmark for the subsequent assembly of other proteins at the promoter. The TATA box is a key component of many promoters used by RNA polymerase II, and it is typically located 25 nucleotides upstream from the transcription start site. Once TFIID has bound to the TATA box, the other factors assemble, along with RNA polymerase II, to form a complete transcription initiation complex. Although Figure 7-12 shows the general transcription factors piling onto the promoter in a certain order, the actual order of assembly probably differs from one promoter to the next. After RNA polymerase II has been positioned on the promoter, it must be released from the complex of general transcription factors to begin its task of making an RNA molecule. A key step in liberating the RNA polymer- ase is the addition of phosphate groups to its "tail" (see Figure 7-12E). This liberation is initiated by the general transcription factor TFIIH, which contains a protein kinase as one of its subunits. Once transcription has begun, most of the general transcription factors dissociate from the DNA and then are available to initiate another round of transcription with a new RNA polymerase molecule. When RNA polymerase II nishes tran- scribing a gene, it too is released from the DNA; the phosphates on its tail are stripped off by protein phosphatases, and the polymerase is then ready to nd a new promoter. Only the dephosphorylated form of RNA polymerase II can initiate RNA synthesis.

start codon mechanism bacteria

The mechanism for selecting a start codon is different in bacteria. Bacterial mRNAs have no 5′ caps to tell the ribosome where to begin searching for the start of translation. Instead, they contain speci c ribosome-binding sequences, up to six nucleotides long, that are located a few nucleotides upstream of the AUGs at which translation is to begin. Unlike a eukaryo- tic ribosome, a prokaryotic ribosome can readily bind directly to a start codon that lies in the interior of an mRNA, as long as a ribosome-binding site precedes it by several nucleotides. Such ribosome-binding sequences are necessary in bacteria, as prokaryotic mRNAs are often polycistronic— that is, they encode several different proteins, each of which is translated from the same mRNA molecule (Figure 7-37). In contrast, a eukaryotic mRNA usually carries the information for a single protein.

leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

ribosomal complex

The recognition of a codon by the anticodon on a tRNA molecule depends on the same type of complementary base-pairing used in DNA replication and transcription. However, accurate and rapid translation of mRNA into protein requires a molecular machine that can move along the mRNA, capture complementary tRNA molecules, hold the tRNAs in position, and then covalently link the amino acids that they carry to form a polypeptide chain. In both prokaryotes and eukaryotes, the machine that gets the job done is the ribosome—a large complex made from dozens of small pro- teins (the ribosomal proteins) and several crucial RNA molecules called ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm.

Exons

Most pro- tein-coding eukaryotic genes, in contrast, have their coding sequences interrupted by long, noncoding, intervening sequences called introns. The scattered pieces of coding sequence—called expressed sequences or exons—are usually shorter than the introns, and they often represent only a small fraction of the total length of the gene (Figure 7-17). Introns range in length from a single nucleotide to more than 10,000 nucleotides. Some protein-coding eukaryotic genes lack introns altogether, and some have only a few; but most have many (Figure 7-18). Note that the terms "exon" and "intron" apply to both the DNA and the corresponding RNA sequences.

Polyadenylation

Polyadenylation provides a newly transcribed mRNA with a spe- cial structure at its 3′ end. In contrast with bacteria, where the 3′ end of an mRNA is simply the end of the chain synthesized by the RNA polymerase, the 3′ end of a forming eukaryotic mRNA is rst trimmed by an enzyme that cuts the RNA chain at a particular sequence of nucleotides. The transcript is then nished off by a second enzyme that adds a series of repeated adenine (A) nucleotides to the cut end. This poly-A tail is generally a few hundred nucleotides long (see Figure 7-16A).

Translation

Process by which mRNA is decoded and a protein is produced,--The conversion of the information in RNA into protein rep- resents a translation of the information into another language that uses different symbols. Because there are only 4 different nucleotides in mRNA but 20 different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein.

RNA capping

RNA capping modifes the 5′ end of the RNA transcript, the end that is synthesized first. The RNA is capped by the addition of an atypical nucleotide—a guanine (G) nucleotide bearing a methyl group, which is attached to the 5′ end of the RNA in an unusual way (Figure 7-16). This capping occurs after RNA polymerase II has produced about 25 nucleotides of RNA, long before it has completed transcribing the whole gene.

protein domain

RNA splicing also provides another advantage to eukaryotes, one that is likely to have been profoundly important in the early evolutionary history of genes. As we discuss in detail in Chapter 9, the intron-exon structure of genes is thought to have sped up the emergence of new and useful proteins: novel proteins appear to have arisen by the mixing and match- ing of different exons of preexisting genes, much like the assembly of a new type of machine from a kit of preexisting functional components. Indeed, many proteins in present-day cells resemble patchworks com- posed from a common set of protein pieces, called protein domains (see Figure 4-51).

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins. The intron-exon type of gene arrangement in eukaryotes may, at rst, seem wasteful. It does, however, have a number of important bene ts. First, the transcripts of many eukaryotic genes can be spliced in differ- ent ways, each of which can produce a distinct protein. Such alternative splicing thereby allows many different proteins to be produced from the same gene (Figure 7-22). About 95% of human genes are thought to undergo alternative splicing. Thus RNA splicing enables eukaryotes to increase the already enormous coding potential of their genomes.

replication fork

a Y-shaped point that results when the two strands of a DNA double helix separate so that the DNA molecule can be replicated. Two are formed at the same time forming a "bubble"--- The two forks move away from the origin in opposite directions, unzipping the DNA double helix and replicating the DNA as they go (Figure 6-9). DNA replication in bacterial and eukaryotic chromosomes is therefore termed bidirectional. The forks move very rapidly—at about 1000 nucle- otide pairs per second in bacteria and 100 nucleotide pairs per second in humans. The slower rate of fork movement in humans (indeed, in all eukaryotes) may be due to the dif culties in replicating DNA through the more complex chromatin structure of eukaryotic chromosomes.

genetic code

collection of codons of mRNA, each of which directs the incorporation of a particular amino acid into a protein during protein synthesis--The rules by which the nucleotide sequence of a gene, through an intermediary mRNA molecule, is translated into the amino acid sequence of a protein are known as the genetic code.

replication machine

complex involving dozens of different enzymes and other proteins that work closely together in the process of DNA replication and interact at the replication fork

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.---For the leading strand, an RNA primer is needed only to start replica- tion at a replication origin; once a replication fork has been established, the DNA polymerase is continuously presented with a base-paired 3ʹ end as it tracks along the template strand. But on the lagging strand, where DNA synthesis is discontinuous, new primers are needed to keep polymerization going (see Figure 6-13). The movement of the replication fork continually exposes unpaired bases on the lagging strand template, and new RNA primers are laid down at intervals along the newly exposed, single-stranded stretch. DNA polymerase adds a deoxyribonucleotide to the 3ʹ end of each primer to start a new Okazaki fragment, and it will continue to elongate this fragment until it runs into the next RNA primer (Figure 6-17).

telemorase

A serious problem arises, however, as the replication fork approaches the end of a chromo- some: although the leading strand can be replicated all the way to the chromosome tip, the lagging strand cannot. When the nal RNA primer on the lagging strand is removed, there is no way to replace it (Figure 6-21). Without a strategy to deal with this problem, the lagging strand would become shorter with each round of DNA replication; after repeated cell divisions, chromosomes would shrink—and eventually lose valuable genetic information. Bacteria solve this "end-replication" problem by having circular DNA molecules as chromosomes. Eukaryotes solve it by having long, repeti- tive nucleotide sequences at the ends of their chromosomes which are incorporated into structures called telomeres. These telomeric DNA sequences attract an enzyme called telomerase to the chromosome ends. Using an RNA template that is part of the enzyme itself, telomerase extends the ends of the replicating lagging strand by adding multiple cop- ies of the same short DNA sequence to the template strand. This extended template allows replication of the lagging strand to be completed by con- ventional DNA replication (Figure 6-22).

RNA polymerases can start an RNA chain without a primer. ---makes one mistake in 10^4 nucleotides copied.

Although RNA polymerase catalyzes essentially the same chemical reac- tion as DNA polymerase, there are some important differences between the two enzymes. First, and most obviously, RNA polymerase uses ribo- nucleoside for phosphates as substrates, so it catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA polymerase involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference likely evolved because tran- scription need not be as accurate as DNA replication; unlike DNA, RNA is not used as the permanent storage form of genetic information in cells, so mistakes in RNA transcripts have relatively minor consequences for a cell. RNA polymerases make about one mistake for every 10^4 nucleotides copied into RNA, whereas DNA polymerase makes only one mistake for every 10^7 nucleotides copied.

mismatch repair

Although the high delity and proofreading abilities of the cell's replica- tion machinery generally prevent replication errors from occurring, rare mistakes do happen. Fortunately, the cell has a backup system—called mismatch repair—which is dedicated to correcting these errors. The replication machine makes approximately one mistake per 107 nucle- otides copied; DNA mismatch repair corrects 99% of these replication errors, increasing the overall accuracy to one mistake in 109 nucleotides copied. This level of accuracy is much, much higher than that generally encountered in our day-to-day lives (table 6-1). Whenever the replication machinery makes a copying mistake, it leaves behind a mispaired nucleotide (commonly called a mismatch). If left uncorrected, the mismatch will result in a permanent mutation in the next round of DNA replication (Figure 6-27). A complex of mismatch repair proteins recognizes such a DNA mismatch, removes a portion of the DNA strand containing the error, and then resynthesizes the missing DNA. This repair mechanism restores the correct sequence (Figure 6-28). To be effective, the mismatch repair system must be able to recognize which of the DNA strands contains the error. Removing a segment from the strand of DNA that contains the correct sequence would only compound the mistake. The way the mismatch system solves this prob- lem is by always removing a portion of the newly made DNA strand. In bacteria, newly synthesized DNA lacks a type of chemical modi cation that is present on the preexisting parent DNA. Other cells use other strat- egies for distinguishing their parent DNA from a newly replicated strand.---Eukaryotes---newly synthesized strand contains nick which mismatch repair protein recognizes as belonging to the newly synth. strand.

RNA processing---Eukaryotic mRNAs are processed in the nucleus.

Although the templating principle by which DNA is transcribed into RNA is the same in all organisms, the way in which the RNA transcripts are handled before they can be used by the cell to make protein differs greatly between bacteria and eukaryotes. Bacterial DNA lies directly exposed to the cytoplasm, which contains the ribosomes on which protein syn- thesis takes place. As an mRNA molecule in a bacterium starts to be synthesized, ribosomes immediately attach to the free 5′ end of the RNA transcript and begin translating it into protein. In eukaryotic cells, by contrast, DNA is enclosed within the nucleus. Transcription takes place in the nucleus, but protein synthesis takes place on ribosomes in the cytoplasm. So, before a eukaryotic mRNA can be translated into protein, it must be transported out of the nucleus through small pores in the nuclear envelope (Figure 7-14). Before it can be exported to the cytosol, however, a eukaryotic RNA must go through several RNA processing steps, which include capping, splicing, and poly- adenylation, as we discuss shortly. These steps take place as the RNA is being synthesized. The enzymes responsible for RNA processing ride on the phosphorylated tail of eukaryotic RNA polymerase II as it synthesizes an RNA molecule (see Figure 7-12), and they process the transcript as it emerges from the polymerase (Figure 7-15).

DNA proofreading and repair

DNA polymerase contains separate sites for DNA synthesis and proofreading.----when DNA polymerase makes a rare mistake and adds the wrong nucle- otide, it can correct the error through an activity called proofreading. Proofreading takes place at the same time as DNA synthesis. Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand. If so, the polymerase adds the next nucleotide; if not, the polymerase clips off the mispaired nucleotide and tries again (Figure 6-14). This proofreading is carried out by a nuclease that cleaves the phosphodiester backbone. Polymerization and proofreading are tightly coordinated, and the two reactions are carried out by different catalytic domains in the same polymerase molecule (Figure 6-15). This proofreading mechanism explains why DNA polymerases synthesize DNA only in the 5ʹ-to-3ʹ direction, despite the need that this imposes for a cumbersome backstitching mechanism at the replication fork (see Figure 6-13). A hypothetical DNA polymerase that synthesized in the 3ʹ-to-5ʹ direction (and would thereby circumvent the need for backstitching) would be unable to proofread: if it removed an incorrectly paired nucle- otide, the polymerase would create a chemical dead end—a chain that could no longer be elongated. Thus, for a DNA polymerase to function as a self-correcting enzyme that removes its own polymerization errors as it moves along the DNA, it must proceed only in the 5ʹ-to-3ʹ direction.

semiconservative replication

DNa replication is "semiconservative" because each daughter DNa double helix is composed of one conserved strand and one newly synthesized strand.

mRNA modifications in eukaryotes---in the nucleus--

Different types of RNA are processed in different ways before leaving the nucleus. Two processing steps, capping and polyadenylation, occur only on RNA transcripts destined to become mRNA molecules (called precur- sor mRNAs, or pre-mRNAs). These two modfications—capping and polyadenylation—increase the stability of a eukaryotic mRNA molecule, facilitate its export from the nucleus to the cytoplasm, and generally mark the RNA molecule as an mRNA. They are also used by the protein-synthesis machinery to make sure that both ends of the mRNA are present and that the message is therefore complete before protein synthesis begins.

aminoacyl-tRNA synthetase

For a tRNA molecule to carry out its role as an adaptor, it must be linked— or charged—with the correct amino acid. How does each tRNA molecule recognize the one amino acid in 20 that is its right partner? Recognition and attachment of the correct amino acid depend on enzymes called aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. In most organisms, there is a different synthetase enzyme for each amino acid. That means that there are 20 synthetases in all: one attaches glycine to all tRNAs that recog- nize codons for glycine, another attaches phenylalanine to all tRNAs that recognize codons for phenylalanine, and so on. Each synthetase enzyme recognizes speci c nucleotides in both the anticodon and the amino- acid-accepting arm of the correct tRNA (Movie 7.6). The synthetases are thus equal in importance to the tRNAs in the decoding process, because it is the combined action of the synthetases and tRNAs that allows each codon in the mRNA molecule to specify its proper amino acid. The synthetase-catalyzed reaction that attaches the amino acid to the 3′ end of the tRNA is one of many reactions in cells coupled to the energy- releasing hydrolysis of ATP (see Figure 3-33). The reaction produces a high-energy bond between the charged tRNA and the amino acid. The energy of this bond is later used to link the amino acid covalently to the growing polypeptide chain.

Ribosomes function

How does the ribosome choreograph all the movements required for translation? In addition to a binding site for an mRNA molecule, each ribosome contains three binding sites for tRNA molecules, called the A site, the P site, and the E site (Figure 7-33). To add an amino acid to a growing peptide chain, the appropriate charged tRNA enters the A site by base-pairing with the complementary codon on the mRNA molecule. Its amino acid is then linked to the peptide chain held by the tRNA in the neighboring P site. Next, the large ribosomal subunit shifts forward, mov- ing the spent tRNA to the E site before ejecting it (Figure 7-34). This cycle of reactions is repeated each time an amino acid is added to the polypep- tide chain, with the new protein growing from its amino to its carboxyl end until a stop codon in the mRNA is encountered.

Nuclear Pore Complex (NPC)

The remaining RNA fragments— excised introns, broken RNAs, and aberrantly spliced transcripts—are not only useless, but they could be dangerous to the cell if allowed to leave the nucleus. How, then, does the cell distinguish between the relatively rare mature mRNA molecules it needs to export to the cytosol and the overwhelming amount of debris generated by RNA processing? The answer is that the transport of mRNA from the nucleus to the cytosol, where mRNAs are translated into protein, is highly selective: only correctly processed mRNAs are exported. This selective transport is mediated by nuclear pore complexes, which connect the nucleoplasm with the cytosol and act as gates that control which macromolecules can enter or leave the nucleus (discussed in Chapter 15). To be "export ready," an mRNA molecule must be bound to an appropriate set of proteins, each of which recognizes different parts of a mature mRNA molecule. These proteins include poly-A-binding proteins, a cap-binding complex, and proteins that bind to mRNAs that have been appropriately spliced (Figure 7-23). The entire set of bound proteins, rather than any single protein, ultimately determines whether an mRNA molecule will leave the nucleus. The "waste RNAs" that remain behind in the nucleus are degraded there, and their nucleotide building blocks are reused for transcription.

Transcription

The rst step a cell takes in expressing one of its many thousands of genes is to copy the nucleotide sequence of that gene into RNA. The proc- ess is called transcription because the information, though copied into another chemical form, is still written in essentially the same language— the language of nucleotides. All the RNA in a cell is made by transcription, a process that has certain similarities to DNA replication (discussed in Chapter 6). Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthe- sis of RNA. Ribonucleotides are added, one by one, to the growing RNA chain; as in DNA replication, the nucleotide sequence of the RNA chain is determined by complementary base-pairing with the DNA template. When a good match is made, the incoming ribonucleotide is covalently linked to the growing RNA chain by the enzyme RNA polymerase. The RNA chain produced by transcription—the RNA transcript—is therefore elongated one nucleotide at a time and has a nucleotide sequence exactly complementary to the strand of DNA used as the template. Transcription differs from DNA replication in several crucial respects. Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is dis- placed and the DNA helix re-forms. For this reason—and because only one strand of the DNA molecule is transcribed—RNA molecules are single-stranded. Further, because RNAs are copied from only a limited region of DNA, RNA molecules are much shorter than DNA molecules; DNA molecules in a human chromosome can be up to 250 million nucle- otide pairs long, whereas most mature RNAs are no more than a few thousand nucleotides long, and many are much shorter than that.

initiator tRNA

The translation of an mRNA begins with the codon AUG, and a special charged tRNA is required to initiate translation. This initiator tRNA always carries the amino acid methionine (or a modi ed form of methio- nine, formyl-methionine, in bacteria). Thus newly made proteins all have methionine as the rst amino acid at their N-terminal end, the end of a protein that is synthesized rst. This methionine is usually removed later by a speci c protease.

mRNA (messenger RNA)

The vast majority of genes carried in a cell's DNA specify the amino acid sequences of proteins. The RNA molecules encoded by these genes—which ultimately direct the synthesis of proteins—are called messenger RNAs (mRNAs). In eukaryotes, each mRNA typically carries information tran- scribed from just one gene, which codes for a single protein; in bacteria, a set of adjacent genes is often transcribed as a single mRNA, which there- fore carries the information for several different proteins

Lagging strand synthesis/completion

To produce a continuous new DNA strand from the many separate pieces of nucleic acid made on the lagging strand, three additional enzymes are needed. These act quickly to remove the RNA primer, replace it with DNA, and join the DNA fragments together. Thus, a nuclease degrades the RNA primer, a DNA polymerase called a repair polymerase then replaces this RNA with DNA (using the end of the adjacent Okazaki fragment as a primer), and the enzyme DNA ligase joins the 5ʹ-phosphate end of one DNA fragment to the adjacent 3ʹ-hydroxyl end of the next (Figure 6-18). Primase can begin new polynucleotide chains, but this activity is possible because the enzyme does not proofread its work. As a result, primers fre- quently contain mistakes. But because primers are made of RNA instead of DNA, they stand out as "suspect copy" to be automatically removed and replaced by DNA. The repair DNA polymerases that make this DNA, like the replicative polymerases, proofread as they synthesize. In this way, the cell's replication machinery is able to begin new DNA chains and, at the same time, ensure that all of the DNA is copied faithfully.

RNA splicing

To produce an mRNA in a eukaryotic cell, the entire length of the gene, introns as well as exons, is transcribed into RNA. After capping, and as RNA polymerase II continues to transcribe the gene, the process of RNA splicing begins, in which the introns are removed from the newly synthe- sized RNA and the exons are stitched together. Each transcript ultimately receives a poly-A tail; in some cases, this happens after splicing, whereas in other cases, it occurs before the nal splicing reactions have been completed. Once a transcript has been spliced and its 5′ and 3′ ends have been modi ed, the RNA is now a functional mRNA molecule that can leave the nucleus and be translated into protein. How does the cell determine which parts of the RNA transcript to remove during splicing? Unlike the coding sequence of an exon, most of the nucleotide sequence of an intron is unimportant. Although there is lit- tle overall resemblance between the nucleotide sequences of different introns, each intron contains a few short nucleotide sequences that act as cues for its removal from the pre-mRNA. These special sequences are found at or near each end of the intron and are the same or very similar in all introns (Figure 7-19). Guided by these sequences, an elaborate splic- ing machine cuts out the intron in the form of a "lariat" structure (Figure 7-20), formed by the reaction of the "A" nucleotide highlighted in red in Figures 7-19 and 7-20.

Anticodon

Two regions of unpaired nucleotides situated at either end of the L-shaped tRNA molecule are crucial to the function of tRNAs in protein synthesis. One of these regions forms the anticodon, a set of three consecutive nucleotides that bind, through base-pairing, to the complementary codon in an mRNA molecule.

stop codons

UAA, UAG, UGA

Primase (RNA polymerase)

We have seen that the accuracy of DNA replication depends on the requirement of the DNA polymerase for a correctly base-paired 3ʹ end before it can add more nucleotides to a growing DNA strand. How then can the polymerase begin a completely new DNA strand? To get the process started, a different enzyme is needed—one that can begin a new polynucleotide strand simply by joining two nucleotides together without the need for a base-paired end. This enzyme does not, however, syn- thesize DNA. It makes a short length of a closely related type of nucleic acid—RNA (ribonucleic acid)—using the DNA strand as a template. This short length of RNA, about 10 nucleotides long, is base-paired to the tem- plate strand and provides a base-paired 3ʹ end as a starting point for DNA polymerase. It thus serves as a primer for DNA synthesis, and the enzyme that synthesizes the RNA primer is known as primase. Primase is an example of an RNA polymerase, an enzyme that synthesizes RNA using DNA as a template. A strand of RNA is very similar chemically to a single strand of DNA except that it is made of ribonucleotide subu- nits, in which the sugar is ribose, not deoxyribose; RNA also differs from DNA in that it contains the base uracil (U) instead of thymine (T) (see Panel 2-6, pp. 76-77). However, because U can form a base pair with A, the RNA primer is synthesized on the DNA strand by complementary base-pairing in exactly the same way as is DNA

nonhomologous end joining

What happens when both strands of the double helix are damaged at the same time? Radiation, mishaps at the replication fork, and vari- ous chemical assaults can all fracture the backbone of DNA, creating a double-strand break. Such lesions are particularly dangerous, because ECB4 m5.51/6.31 they can lead to the fragmentation of chromosomes and the subsequent loss of genes. This type of damage is especially dif cult to repair. Each chromosome contains unique information; if a chromosome undergoes a double- strand break, and the broken pieces become separated, the cell has no spare copy it can use to reconstruct the information that is now missing. To handle this potentially disastrous type of DNA damage, cells have evolved two basic strategies. The rst involves rapidly sticking the broken ends back together, before the DNA fragments drift apart and get lost. This repair mechanism, called nonhomologous end joining, occurs in many cell types and is carried out by a specialized group of enzymes that "clean" the broken ends and rejoin them by DNA ligation. This "quick and dirty" mechanism rapidly repairs the damage, but it comes with a price: in "cleaning" the break to make it ready for ligation, nucleotides are often lost at the site of repair (Figure 6-29A). In most cases, this emergency repair mechanism mends the damage without creating any additional problems. But if the imperfect repair dis- rupts the activity of a gene, the cell could suffer serious consequences. Thus, nonhomologous end joining can be a risky strategy for xing broken chromosomes.

initiation of transcription (bacteria)

When an RNA polymerase collides randomly with a DNA molecule, the enzyme sticks weakly to the double helix and then slides rapidly along its length. RNA polymerase latches on tightly only after it has encountered a gene region called a promoter, which contains a speci c sequence of nucleotides that lies immediately upstream of the starting point for RNA synthesis. Once bound tightly to this sequence, the RNA polymerase opens up the double helix immediately in front of the promoter to expose the nucleotides on each strand of a short stretch of DNA. One of the two exposed DNA strands then acts as a template for complementary base- pairing with incoming ribonucleoside triphosphates, two of which are joined together by the polymerase to begin synthesis of the RNA chain. Chain elongation then continues until the enzyme encounters a second signal in the DNA, the terminator (or stop site), where the polymerase halts and releases both the DNA template and the newly made RNA tran- script (Figure 7-9). This terminator sequence is contained within the gene and is transcribed into the 3ʹ end of the newly made RNA. Because the polymerase must bind tightly before transcription can begin, a segment of DNA will be transcribed only if it is preceded by a promoter. This ensures that those portions of a DNA molecule that contain a gene will be transcribed into RNA.

Promoter

specific region of a gene where RNA polymerase can bind and begin transcription---The next problem an RNA polymerase faces is determining which of the two DNA strands to use as a template for transcription: each strand has a different nucleotide sequence and would produce a different RNA transcript. The secret lies in the structure of the promoter itself. Every promoter has a certain polarity: it contains two different nucleotide sequences upstream of the transcriptional start site that position the RNA polymerase, ensuring that it binds to the promoter in only one orientation. Because the polymerase can only synthesize RNA in the 5′-to-3′ direction once the enzyme is bound it must use the DNA strand oriented in the 3′-to-5′ direction as its template. This selection of a template strand does not mean that on a given chro- mosome, transcription always proceeds in the same direction. With respect to the chromosome as a whole, the direction of transcription var- ies from gene to gene. But because each gene typically has only one promoter, the orientation of its promoter determines in which direction that gene is transcribed and therefore which strand is the template strand (Figure 7-11).

eukaryotic ribosomes

the eukaryotic ribosome is a large complex of four rRnAs and more than 80 small proteins. prokaryotic ribosomes are very similar: both are formed from a large and small subunit, which only come together after the small subunit has bound an mrNa. although ribosomal proteins greatly outnumber rrNas, the rNas account for most of the mass of the ribosome and give it its overall shape and structure.

Depurination

the loss of a purine base from a nucleotide

5' to 3' direction

the only direction that DNA polymerase can synthesize DNA; it does so by adding nucleotides to the 3' end of a DNA strand.