Cell Biology Ch.7

How do eukaryotes handle their RNA transcripts?

(A) In eukaryotic cells, the pre-mRNA molecule produced by transcription contains both intron and exon sequences. Its two ends are modified, and the introns are removed by RNA splicing. The resulting mRNA is then transported from the nucleus to the cytoplasm, where it is translated into protein. Although these steps are depicted as occurring in sequence, one at a time, in reality they occur simultaneously. For example, the RNA cap is usually added and splicing usually begins before transcription has been completed. Because of this overlap, transcripts of the entire gene (including all introns and exons) do not typically exist in the cell.

What is a TATA box?

A TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins. The TATA box is named for its conserved DNA sequence, which is most commonly TATAAA.

Describe the overall structural differences between DNA and RNA, but also why RNA can take on many different structural complexes that DNA cannot.

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; double- stranded DNA cannot fold in this fashion.

What is the significance of polyadenlyation and capping?

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 precursor mRNAs, or pre-mRNAs). • These two modifications—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.

What dictates how the chain will fold to form a molecule with a distinctive shape and chemistry within a protein?

Each type of protein has its own unique amino acid sequence, which dictates how the chain will fold to form a molecule with a distinctive shape and chemistry. The genetic instructions carried by DNA must therefore specify the amino acid sequences of proteins.

Describe RNA splicing and what introns and exons are.

Eukaryotic mRNA processing also involves RNA splicing • Eukaryotic genes are often interrupted by noncoding sequences (introns). Need to remove/splice these introns out to get finished/meaningful message. • Exons-expressed sequences • Introns-intervening/nonexpressed sequences

How do proteasomes select which proteins in the cell should be degraded?

In eukaryotes, proteasomes act primarily on proteins that have been marked for destruction by the covalent attachment of a small protein called ubiquitin. Specialized enzymes tag selected proteins with a short chain of ubiquitin molecules; these ubiquitylated proteins are then recognized, unfolded, and fed into proteasomes by proteins in the stopper. • Proteins that are meant to be short-lived often contain a short amino acid sequence that identifies the protein as one to be ubiquitylated and degraded in proteasomes. Damaged or misfolded proteins, as well as proteins containing oxidized or otherwise abnormal amino acids, are also recognized and degraded by this ubiquitin-dependent proteolytic system. The enzymes that add a polyubiquitin chain to such proteins rec- ognize signals that become exposed on these proteins as a result of the misfolding or chemical damage—for example, amino acid sequences or conformational motifs that remain buried and inaccessible in the normal "healthy" protein.

Where are eukaryotic mRNAs process?

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

How are introns removed from Pre-mRNAs?

Introns Are Removed From Pre-mRNAs by 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 • The introns are removed from the newly synthesized 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 final splicing reactions have been completed. Once a transcript has been spliced and its 5 and 3 ends have been modified, the RNA is now a functional mRNA molecule that can leave the nucleus and be translated into protein.

What are the steps RNA polymerase must undergo to have RNA processing proteins to assemble on its tail?

Phosphorylation of RNA polymerase II allows RNA processing proteins to assemble on its tail 1. Polymerase transcribes DNA into RNA. 2. Polymerase also carries RNA-processing proteins that act on newly formed RNA. 3. Proteins bind to RNA polymerase tail when it is phosphorylated late in the process of transcription initiation. 4. Capping, polyadenylation, and splicing are modification made to RNA during this process.

What translates RNA to proteins?

Proteins are translated by polyribosomes • A series of ribosomes can simultaneously translate the same mRNA molecule. • EM of polyribosome/polysomes. • New ribosome hops onto the 5' end of mRNA molecule before first has completed translation. • Many protein molecules can be made in a given time.

How does RNA capping work?

RNA capping modifies 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 This capping occurs after RNA polymerase II has produced about 25 nucleotides of RNA, long before it has completed transcribing the whole gene

How is RNA splicing carried out?

RNA splicing is carried out largely by RNA molecules rather than proteins. • These RNA molecules, called small nuclear RNAs (snRNAs), are packaged with additional proteins to form small nuclear ribonucleoproteins (snRNPs, pronounced "snurps"). • The snRNPs recognize splice-site sequences through complementary base-pairing between their RNA components and the sequences in the pre-mRNA, and they also participate intimately in the chemistry of splicing • Together, these snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that carries out RNA splicing in the nucleus.

How do ribosomes where to start and stop translation?

Specific Codons in mRNA Signal the Ribosome Where to Start and to Stop Protein Synthesis In a cell, however, a specific start signal is required to initiate translation. The site at which protein synthesis begins on an mRNA is crucial, because it sets the reading frame for the whole length of the message. An error of one nucleotide either way at this stage will cause every subsequent codon in the mRNA to be misread, resulting in a nonfunctional protein with a garbled sequence of amino acids. And the rate of initiation determines the rate at which the protein is synthesized from the mRNA. • Initiator tRNA coupled to methionine is loaded on small ribosomal subunit + proteins = translation initiation factors. • Only charged initiator tRNA is capable of binding tightly to P-site of small ribosomal subunit. • It then binds to the 5' end of an mRNA, signaled by cap present mRNA. • It moves forward (5' to 3') along mRNA searching for first AUG. • Several initiation factors dissociate from small ribosomal subunit to make way for large ribosomal subunit to assemble. • Protein synthesis begins with the addition of the next charged tRNA to A-site. • 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 modified form of methionine, formyl-methionine, in bacteria). Thus newly made proteins all have methionine as the first amino acid at their N-terminal end, the end of a protein that is synthesized first. This methionine is usually removed later by a specific protease. • In eukaryotes, an initiator tRNA, charged with methionine, is first loaded into the P site of the small ribosomal subunit, along with additional proteins called translation initiation factors the 5 end of an mRNA molecule, which is marked by the 5 cap that is present on all eukaryotic mRNAs. • The small ribosomal subunit then moves forward (5 to 3 ) along the mRNA searching for the first 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. • 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 specific 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. In contrast, a eukaryotic mRNA usually carries the information for a single protein. o Bacteria use a Shine-Delgarno sequence (~6 bps long) upstream of the AUG to cue the start of translation, while eukaryotes use a Kozak sequence. o Genes organized into clusters (operons) that are translated together with single mRNA. o Bacterial mRNA have no 5' caps to tell ribosome where to begin searching for start of translation. o Contain specific ribosome-binding sequences up to 6 nucleotides long upstream of AUG to begin translation. o Ribosome binding sites can be in interior of mRNA molecule. o This allows them to synthesize several separate proteins from single mRNA molecule.

How is splicing carried out?

Splicing is carried out by a collection of RNA-protein complexes called snRNPs. • Introns are removed by RNA splicing o Alternative splicing leads to greater protein diversity from single gene. o Can produce distinct proteins. o Many proteins from same gene. o ~60% of human genes undergo alternative splicing. o RNA splicing enables increases coding potential of their genomes. • There are five snRNPs, called U1, U2, U4, U5, and U6 1. U1 and U2 bind to the 5' splice site (U1) and the lariat branch point (U2) though complementary base pairing. 2. Additional snRPs are attracted to the splice site, and interactions between their protein components drive the assembly of the complete spliceosome. 3. Rearranements in the base pairs that hold together the snRNPs and the RNA transcript then reorganize the spliceosome to form the active site that excises the intron, leaving the spliced mRNa behind.

What is TBP?

TATA-binding protein (TBP) binds to the TATA box (indicated by letters) and bends the DNA double helix. The unique distortion of DNA caused by TBP, which is a subunit of TFIID helps attract the other general transcription factors. • TBP is a single polypeptide chain that is folded into two very similar domains • The protein sits atop the DNA double helix like a saddle on a bucking horse

In bacteria how does RNA polymerase determine which of the two DNA strands to use as a template for transcription when 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.

How exactly are genes expressed?

Transcription and translation are the means by which cells read out, or express, the instructions in their genes

What does tetracyclin do?

blocks binding of aminoacyl-tRNA to A site of ribosome

What does Rifamycin do?

blocks initiation of transcription by binding to RNA polymerase

What does chloramphenicol do?

blocks the peptidyl transferase reaction on ribosomes

What does ribosomal RNA do?

form the core of the ribosome's structure and catalyze protein synthesis

What does messenger RNA do?

messenger RNAs (mRNAs) code for proteins

What does streptomycin do?

prevents the transition from initiation complex to chain elongation; also causes miscoding

What do microRNAs do?

regulate gene expression

What do other noncoding RNAs do?

used in RNA splicing, gene regulation, telomere maintenance, and many other processes

What does cyclohexamide do?

blocks the translocation reaction on ribosomes

What do transfer RNAs do?

serve as adaptors between mRNA and amino acids during protein synthesis

How does tRNA make

tRNA Molecules Match Amino Acids to Codons in mRNA • The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, 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 transfer RNAs (tRNAs), each about 80 nucleotides in length. • More than 1 tRNA for many AAs. • Some tRNAs recognize > 1 codon (wobble phenomenon. • Unpaired nucleotides: anticodon. • Anticodon bind complementary codon in mRNA 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. The other is a short single-stranded region at the 3 end of the molecule; this is the site where the amino acid that matches the codon is covalently attached to the tRNA.

The chemical structure of RNA differs slightly from that of DNA.

(A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional -OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a -CH3 group. (C) A short length of RNA. The chemical linkage between nucleotides in RNA—a phosphodiester bond—is the same as that in DNA.

How do prokaryotes handle their RNA transcripts?

(B) In prokaryotes, the production of mRNA molecules is simpler. The 5 end of an mRNA molecule is produced by the initiation of transcription by RNA polymerase, and the 3 end is produced by the termination of transcription. Because prokaryotic cells lack a nucleus, transcription and translation take place in a common compartment. Translation of a bacterial mRNA can therefore begin before its synthesis has been completed. In both eukaryotes and prokaryotes, the amount of a protein in a cell depends on the rates of each of these steps, as well as on the rates of degradation of the mRNA and protein molecules.

What are the steps of translation?

1. In step 1, a charged tRNA carrying the next amino acid to be added to the polypeptide chain binds to the vacant A site on the ribosome by forming base pairs with the mRNA codon that is exposed there. Because only the appropriate tRNA molecules can base-pair with each codon, this codon determines the specific amino acid added. The A and P sites are sufficiently close together that their two tRNA molecules are forced to form base pairs with codons that are contiguous, with no stray bases in between. This positioning of the tRNAs ensures that the correct reading frame will be preserved throughout the synthesis of the protein. 2. In step 2, the carboxyl end of the polypeptide chain (amino acid 3 in step 1) is uncoupled from the tRNA at the P site and joined by a peptide bond to the free amino group of the amino acid linked to the tRNA at the A site. This reaction is catalyzed by an enzymatic site in the large subunit. 3. In step 3, a shift of the large subunit relative to the small subunit moves the two tRNAs into the E and P sites of the large subunit. 4. In step 4, the small subunit moves exactly three nucleotides along the mRNA molecule, bringing it back to its original position relative to the large subunit. This movement ejects the spent tRNA and resets the ribosome with an empty A site so that the next charged tRNA molecule can bind. As indicated, the mRNA is translated in the 5 -to-3 direction, and the N-terminal end of a protein is made first, with each cycle adding one amino acid to the C-terminus of the polypeptide chain. To watch the translation cycle in atomic detail, see. • Only two sites occupied at any given time. • tRNA enters A-site by base pairing with codon on mRNA; a.a. linked to peptide chain held by tRNA in P-site; spent tRNA is moved to E-site before being ejected.

How is eukaryotic transcription different from bacterial transcription?

1. The first difference lies in the RNA polymerase themselves. While bacteria contain a single type of RNA polymerase, eukaryotic cells have three—RNA polymerase I, RNA polymerase II, and RNA polymerase III. 2. The second difference is that, whereas the bacterial RNA polymerase (along with its sigma subunit) is able to initiate transcription on its own, eukaryotic RNA polymerases require the assistance of a large set of accessory proteins. Principal among these are the general transcription factors, which must assemble at each promoter, along with the polymerase, before the polymerase can begin transcription. 3. A third distinctive feature of transcription in eukaryotes is that the mechanisms that control its initiation are much more elaborate than those prokaryotes. In bacteria, genes tend to lie very close to one another in the DNA, with only very short lengths of non-transcribed DNA between them. But in plants and animals, including humans, individual genes are spread out along the DNA. This architecture allows a single gene to be controlled by a large variety of regulatory DNA sequences scattered along the DNA, and it enables eukaryotes to engage in more complex forms of transcriptional regulation than do bacteria. 4. Eukaryotic transcription initiation must take into account the packing of DNA into nucleosomes and more compact forms of chromatin structure.

What are the steps in bacterial transcription?

1. 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. 2. RNA polymerase latches on tightly only after it has encountered a gene region called a promoter, which contains a specific sequence of nucleotides that lies immediately upstream of the starting point for RNA synthesis. 3. 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. 4. 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. 5. 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 transcript. This terminator sequence is contained within the gene and is transcribed into the 3 end of the newly made RNA. 6. 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.

What kind of structure does a pre-mRNA molecule form during RNA splicing?

An intron in a pre-mRNA molecule forms a branched structure during RNA splicing. • In the first step, the branch point adenine in the intron sequence attacks the 5' splice site and cuts the sugar-phosphate backbone of the RNA at this point. o In this process, the cut 5' end of the intron becomes covalently linked to the 2'-OH group of the ribose of the A nucleotide to form a branched structure. • The free 3'-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together into a continuous coding sequence and releasing the intron in the form of a lariat structure, which is eventually degraded in the nucleus.

Where are bacterial mRNA processed?

Bacterial DNA lies directly exposed to the cytoplasm, which contains the ribosomes on which protein synthesis 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.

What must eukaryotic mRNA go through before it gets processed?

Before it can be exported to the cytosol, however, a eukaryotic RNA must go through several RNA processing steps, which include capping, splicing, and polyadenylation, 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, and they process the transcript as it emerges from the polymerase

What does controlled protein breakdown help do?

Controlled Protein Breakdown Helps Regulate the Amount of Each Protein in a Cell • After a protein is released from the ribosome, a cell can control its activity and longevity in various ways. The number of copies of a protein in a cell depends, like the human population, not only on how quickly new individuals are made but also on how long they survive. So controlling the breakdown of proteins into their constituent amino acids helps cells regulate the amount of each particular protein. • In eukaryotic cells, proteins are broken down by large protein machines called proteasomes, present in both the cytosol and the nucleus. A proteasome contains a central cylinder formed from proteases whose active sites face into an inner chamber. Each end of the cylinder is stoppered by a large protein complex formed from at least 10 types of protein subunits • These protein stoppers bind the proteins destined for deg- radation and then—using ATP hydrolysis to fuel this activity—unfold the doomed proteins and thread them into the inner chamber of the cylinder. Once the proteins are inside, proteases chop them into short peptides, which are then jettisoned from either end of the proteasome. Housing proteases inside these molecular destruction chambers makes sense, as it prevents the enzymes from running rampant in the cell.

What exactly is a gene and how does DNA synthesize proteins?

DNA does not synthesize proteins itself, but it acts like a manager, delegating the various tasks to a team of workers. • When the cell needs a particular protein, the nucleotide sequence of the appropriate segment of a DNA molecule is first copied into another type of nucleic acid—RNA (ribonucleic acid). o That segment of DNA is called a gene.

What is required for eukaryotic transcription to take place?

Eukaryotic RNA Polymerase Requires General Transcription Factors The initial finding that, unlike bacterial RNA polymerase, purified eukaryotic RNA polymerase II could not initiate transcription on its own in a test tube led to the discovery and purification of the general transcription 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 transcription. • Thus the general transcription factors have a similar role in eukaryotic transcription as sigma factor has in bacterial transcription.

How are eukaryotic and bacterial genes organized differently?

Eukaryotic and bacterial genes are organized differently. A bacterial gene consists of a single stretch of uninterrupted nucleotide sequence that encodes the amino acid sequence of a protein (or more than one protein). In contrast, the protein-coding sequences of most eukaryotic genes (exons) are interrupted by noncoding sequences (introns). Promoters for transcription are indicated in green. • Eukaryotic genes (Exons) are interrupted by noncoding sequences (Introns). • Exons are shorter; Introns are longer: can be up to 10,000 nucleotides. • Some no introns, others few, most have many.

Describe RNA splicing and what introns and exons are.

In Eukaryotes, Protein-Coding Genes Are Interrupted by Noncoding Sequences Called Introns Most eukaryotic pre-mRNAs have to undergo an additional processing step before they are functional mRNAs. This step involves a far more radical modification of the pre-mRNA transcript than capping or polyadenylation, and it is the consequence of a surprising feature of most eukaryotic genes. In bacteria, most proteins are encoded by an uninterrupted stretch of DNA sequence that is transcribed into an mRNA that, without any further processing, can be translated into protein. Most protein-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 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 synthesized 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 final splicing reactions have been completed. Once a transcript has been spliced and its 5 and 3 ends have been modified, the RNA is now a functional mRNA molecule that can leave the nucleus and be translated into protein.

What in bacteria is primarily responsible for recognizing the promoter sequence on the DNA?

In bacteria, it is a subunit of RNA polymerase, the sigma (σ) factor that is primarily responsible for recognizing the promoter sequence on the DNA.

What is a promotor?

In genetics, a promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand).

How can the sigma (σ) "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 find the promoter sequence without having to separate the entwined DNA strands.

Why does life require autocatalysis?

Life Requires Autocatalysis • The origin of life requires molecules that possess, if only to a small extent, one crucial property: the ability to catalyze reactions that lead—directly or indirectly—to the production of more molecules like themselves. Catalysts with this self-producing property, once they had arisen by chance, would divert raw materials from the production of other substances to make more of themselves. In this way, one can envisage the gradual develop- ment of an increasingly complex chemical system of organic monomers and polymers that function together to generate more molecules of the same types, fueled by a supply of simple raw materials in the primitive environment on Earth. Such an autocatalytic system would have many of the properties we think of as characteristic of living matter: the sys- tem would contain a far-from-random selection of interacting molecules; it would tend to reproduce itself; it would compete with other systems dependent on the same raw materials; and, if deprived of its raw mat- erials or maintained at a temperature that upset the balance of reaction rates, it would decay toward chemical equilibrium and "die."

What is RNA polymerase?

Like the DNA polymerase that carries out DNA replication, RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together and form the sugar-phosphate backbone of the RNA chain. • The RNA polymeras moves stepwise along the DNA, unwinding the DNA helix just ahead to expose a new region of the template strand for complementary base-pairing. In this way the growing RNA chain is extended by one nucleotide at a time in the 5'-to-3' direction. o The incoming ribonuceloside triphosphates (ATP, CTP, UTP, and GTP) provide the energy needed to drive the reaction forward.

How can so many RNA molecules and cell be made within the cell at once?

Many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules. This successive amplification enables cells to rapidly synthesize large amounts of protein whenever necessary. At the same time, each gene can be transcribed, and its RNA translated, at different rates, providing the cell with a way to make vast quantities of some proteins and tiny quantities of others. • A cell can change (or regulate) the expression of each of its genes according to the needs of the moment.

Describe the process by which mRNAs are exported and degraded.

Mature Eukaryotic mRNAs Are Exported from the Nucleus • The Nuclear Pore Complex recognizes and exports only completed mRNAs o Transport out of nucleus highly selective. o Nuclear pore complex recognizes completed mRNAs only. o Must be bound to appropriate proteins: signal correct. o Include polyA-binding proteins, cap-binding complex, complete RNA splice protein (EJC: exon junction complex). o Waste mRNAs digested by RNAse and then reused for transcription. o Sequences in the 3'-UTR help determine how much protein synthesized. o Stability of mRNA: 3'-UTR o mRNA with long lifetimes (3'UTR) high levels of proteins: b-globin. • We have seen how eukaryotic pre-mRNA synthesis and processing take place in an orderly fashion within the cell nucleus. However, these events create a special problem for eukaryotic cells: of the total number of pre- mRNA transcripts that are synthesized, only a small fraction—the mature mRNAs—will be useful to the cell. o 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. • 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 o 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. • 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. • mRNA Molecules Are Eventually Degraded in the Cytosol

What are microRNAs?

MicroRNAs (miRNAs), serve as key regulators of eukaryotic gene expression 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 final product of the gene is a protein, gene expression includes both transcription and translation. • When an RNA molecule is the gene's final product, however, gene expression does not require translation.

How exactly does RNA polymerase move to transcribe DNA into RNA?

On an individual chromosome, some genes are transcribed using one DNA strand as a template, and others are transcribed from the other DNA strand. RNA polymerase always moves in the 3 -to-5 direction and the selection of the template strand is determined by the orientation of the promoter at the beginning of each gene. Thus the genes transcribed from left to right use the bottom DNA strand as the template those transcribed from right to left use the top strand as the template.

How does RNA polyadenylation work?

Polyadenylation provides a newly transcribed mRNA with a special 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 first trimmed by an enzyme that cuts the RNA chain at a particular sequence of nucleotides. The transcript is then finished 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

How can RNA store information and catalyze chemical reactions?

RNA Can Both Store Information and Catalyze Chemical Reactions • • Such complementary templating mechanisms lie at the heart of both DNA rep- lication and transcription in modern-day cells. • But the efficient synthesis of polynucleotides by such complementary templating mechanisms also requires catalysts to promote the polymeri- zation reaction: without catalysts, polymer formation is slow, error-prone, and inefficient. Today, nucleotide polymerization is catalyzed by protein enzymes—such as DNA and RNA polymerases. But how could this reac- tion be catalyzed before proteins with the appropriate catalytic ability existed? The beginnings of an answer were obtained in 1982, when it was discovered that RNA molecules themselves can act as catalysts. The unique potential of RNA molecules to act both as information carriers and as catalysts is thought to have enabled them to have a central role in the origin of life. • In present-day cells, RNA is synthesized as a single-stranded molecule, and we have seen that complementary base-pairing can occur between nucleotides in the same chain. This base-pairing, along with noncon- ventional hydrogen bonds, can cause each RNA molecule to fold up in a unique way that is determined by its nucleotide sequence (see Figure 7-5). Such associations produce complex three-dimensional shapes.

How can RNA molecules form intramolecular base pairs and fold into specific structures?

RNA is single-stranded, but it often contains short stretches of nucleotides that can base-pair with complementary sequences found elsewhere on the same molecule. These interactions—along with some nonconventional base-pair interactions (e.g., A-G)—allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides.

What are the three different types of eukaryotic RNA polymerases and what do they transcribe?

RNA polymerase I-- transcribes most rRNA genes RNA polymerase II-- transcribes all protein-coding genes, miRNA genes, plus genes for other noncoding RNAs (e.g., those in spliceosomes) RNA polymerase III-- tRNA genes 5S rRNA gene and genes for many other small RNAs

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.

What are Ribosomal RNAs?

Ribosomal RNAs (rRNAs) form the structural and catalytic core of the ribosomes, which translate mRNAs into protein. • It constitutes the predominant material within the ribosome, which is approximately 60% rRNA and 40% protein by weight, or 3/5 of ribosome mass. Ribosomes contain two major rRNAs and 50 or more proteins. The ribosomal RNAs form two subunits, the large subunit (LSU) and small subunit (SSU). The LSU rRNA acts as a ribozyme, catalyzing peptide bond formation. rRNA sequences are widely used for working out evolutionary relationships among organisms, since they are of ancient origin and are found in all known forms of life.

How does RNA polymerase know where to start and finish transcription?

Signals in DNA Tell RNA Polymerase Where to Start and Finish Transcription The initiation of transcription is an especially critical process because it is the main point at which the cell selects which proteins or RNAs are to be produced. • To begin transcription, RNA polymerase must be able to recognize the start of a gene and bind firmly to the DNA at this site. • The way in which RNA polymerases recognize the transcription start site of a gene differs somewhat between bacteria and eukaryotes. • The numbers above the DNA indicate the positions of nucleotides counting from the first nucleotide transcribed, which is designated +1. The polarity of the promoter orients the polymerase and determines which DNA strand is transcribed. All bacterial promoters contain DNA sequences at -10 and -35 that closely resemble those shown here.

How does the cell know the beginning and the end of an intron?

Special nucleotide sequences in a pre-mRNA transcript signal the beginning and the end of an intron. • The special sequences are recognized primarily by small nuclear ribonucleoproteins (snRNPs), which direct the cleavage of the RNA at the intron-exon borders and catalyze the covalent linkage of the exon sequences. • The distances along the RNA between the three splicing sequences are highly variable; however, the distance between the branch point and the 5 splice junction is typically much longer than that between the 3 splice junction and the branch point. • R: A or G ; Y: C or U ; N: any nucleotide • A: forms branch point of lariat produced in splicing reaction.

Where exactly did introns arise?

The Earliest Cells May Have Had Introns in Their Genes According to one school of thought, early cells—the common ancestors of prokaryotes and eukaryotes—contained introns that were lost in prokaryotes during subsequent evolution. By shedding their introns and adopting a smaller, more streamlined genome, prokaryotes would have been able to reproduce more rapidly and efficiently. Consistent with this idea, simple eukaryotes that reproduce rapidly (some yeasts, for example) have relatively few introns, and these introns are usually much shorter than those found in higher eukaryotes.

How is the ribosome a ribozyme and what is a ribozyme?

The Ribosome Is a Ribozyme • The ribosome is one of the largest and most complex structures in the cell, composed of two-thirds RNA and one-third protein by weight. The deter- mination of the entire three-dimensional structure of its large and small subunits in 2000 was a major triumph of modern biology. The structure confirmed earlier evidence that the rRNAs—not the proteins—are respon- sible for the ribosome's overall structure and its ability to choreograph and catalyze protein synthesis. • The rRNAs are folded into highly compact, precise three-dimensional structures that form the core of the ribosome (Figure 7-35). In marked contrast to the central positioning of the rRNAs, the ribosomal proteins are generally located on the surface, where they fill the gaps and crevices of the folded RNA. The main role of the ribosomal proteins seems to be to help fold and stabilize the RNA core, while permitting the changes in rRNA conformation that are necessary for this RNA to catalyze efficient protein synthesis • Not only are the three tRNA-binding sites (the A, P, and E sites) on the ribosome formed primarily by the rRNAs, but the catalytic site for peptide bond formation is formed by the 23S rRNA of the large subunit; the near- est ribosomal protein is located too far away to make contact with the incoming charged tRNA or with the growing polypeptide chain. The cata- lytic site in this rRNA—a peptidyl transferase—is similar in many respects to that found in some protein enzymes: it is a highly structured pocket that precisely orients the two reactants—the elongating polypeptide and the charged tRNA—thereby greatly increasing the probability of a produc- tive reaction. • RNA molecules that possess catalytic activity are called ribozymes. Later, in the final section of this chapter, we will consider other ribozymes and discuss what the existence of RNA-based catalysis might mean for the early evolution of life on Earth. Here we need only note that there is good reason to suspect that RNA rather than protein molecules served as the first catalysts for living cells. If so, the ribosome, with its catalytic RNA core, could be viewed as a relic of an earlier time in life's history, when cells were run almost entirely by ribozymes.

How is the genetic code translated with synthetase and tRNA?

The genetic code is translated by the cooperation of two adaptors: aminoacyl-tRNA synthetases and tRNAs. Each synthetase couples a particular amino acid to its corresponding tRNAs, a process called charging. The anticodon on the charged tRNA molecule then forms base pairs with the appropriate codon on the mRNA. An error in either the charging step or the binding of the charged tRNA to its codon will cause the wrong amino acid to be incorporated into a protein chain. In the sequence of events shown, the amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA.

Is the intron and exon arrangement in eukaryotes useless?

The intron-exon type of gene arrangement in eukaryotes may, at first, seem wasteful. It does, however, have a number of important benefits. • First, the transcripts of many eukaryotic genes can be spliced in different ways, each of which can produce a distinct protein. Such alternative splicing thereby allows many different proteins to be produced from the same gene. • RNA splicing also provides another advantage to eukaryotes, one that is likely to have been profoundly important in the early evolutionary history of genes. o 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 matching of different exons of preexisting genes, much like the assembly of a new type of machine from a kit of preexisting functional components.

How and where is the mRNA message decoded?

The mRNA Message Is Decoded by Ribosomes • 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 • 80 ribosomal proteins; several RNA molecules (ribosomal RNA) form ribosome. • Eukaryotic and prokaryotic ribosome very different. • One large one small subunit: together form ribosome. • Two subunits come together on mRNA molecule, near 5'end to begin protein synthesis. • As mRNA moves through it, ribosome translates nucleotide sequence into a.a. sequence one codon at a time using tRNAs as adaptors. • 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.

How is protein made from mRNA?

The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code. Translation is the conversion of RNA into proteins • Genetic code: nucleotide sequence translated into a.a. sequence of protein. • Read consecutively in groups of three. • RNA linear polymer of 4 different nucleotides: 4x4x4 = 64 possible combinations of three nucleotides: AAA, AUA, AUG etc. • Each group of 3 consecutive nucleotides in RNA is called codon. • Each codon specifies an amino acid. • Each a.a. is specified by more than one triplet - code is redundant.

What are mRNAs?

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 transcribed 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 therefore carries the information for several different proteins. • The final product of other genes, however, is the RNA itself. As we see later, these non-messenger RNAs, like proteins, have various roles, serving as regulatory, structural, and catalytic components of cells. They play key parts, for example, in translating the genetic message into protein

What is required by polymerase II to being transcription?

To begin transcription, eukaryotic RNA polymerase II requires a set of general transcription factors. These transcription factors are called TFIIB, TFIID, and so on. (A) Many eukaryotic promoters contain a DNA sequence called the TATA box. (B) The TATA box is recognized by a subunit of the general transcription factor TFIID, called the TATA-binding protein (TBP). (C) The binding of TFIID enables the adjacent binding of TFIIB. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then pries apart the double helix at the transcription start point, using the energy of ATP hydrolysis, which exposes the template strand of the gene (not shown). TFIIH also phosphorylates RNA polymerase II, releasing the polymerase from most of the general transcription factors, so it can begin transcription. The site of phosphorylation is a long polypeptide "tail" that extends from the polymerase.

What are transfer RNAs?

Transfer RNAs (tRNAs) act as adaptors that select specific amino acids and hold them in place on a ribosome for their incorporation into protein. • Is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins. tRNA does this by carrying an amino acid to the protein synthetic machinery of a cell (ribosome) as directed by a three-nucleotide sequence (codon) in a messenger RNA (mRNA). As such, tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code. • tRNA form adaptors that select amino acids and hold them in place on ribosomes for incorporation into proteins

Where does translation stop?

Translation halts at a stop codon. In the final phase of protein synthesis, the binding of release factor to an A site bearing a stop codon terminates translation of an mRNA molecule. The completed polypeptide is released, and the ribosome dissociates into its two separate subunits. • The end of translation in both prokaryotes and eukaryotes is signaled by the presence of one of several codons, called stop codons, in the mRNA. The stop codons—UAA, UAG, and UGA—are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. • Proteins known as release factors bind to any stop codon that reaches the A site on the ribosome; this binding alters the activity of the peptidyl transferase in the ribosome, causing it to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA • This reaction frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule; because this is the only attachment that holds the growing polypeptide to the ribosome, the completed protein chain is immediately released. At this point, the ribosome also releases the mRNA and dissociates into its two separate subunits, which can then assemble on another mRNA molecule to begin a new round of protein synthesis.

What is central dogma?

• The central dogma—that DNA makes RNA that makes protein—presented evolutionary biologists with a knotty puzzle: if nucleic acids are required to direct the synthesis of proteins, and proteins are required to synthesize nucleic acids, how could this system of interdependent components have arisen? One view is that an RNA world existed on Earth before cells containing DNA and proteins appeared. According to this hypothesis, RNA—which today serves largely as an intermediate between genes and proteins—both stored genetic information and catalyzed chemical reactions in primitive cells. Only later in evolutionary time did DNA take over as the genetic material and proteins become the major catalysts and structural components of cells