Fund A Lecture 9: RNA & Protein Synthesis

What is the role of Class II eukaryotic RNA polymerase?

mRNA is first made as a precursor species referred to as *heterogeneous RNA (hnRNA)*, which is made by RNA polymerase II

What are the major inhibitors of prokaryotic protein synthesis?

(1) With initiation, we have AUG on the mRNA coding for methionine, which is the start codon. The antiparallel complementary bases are CAU so the anticodon on tRNA base pairs with the codon on mRNA. UUU is the first triplet codon identified for phenylalanine. Thus, the anticodon on tRNA will have to be AAA to bring in the phenylalanine. Initiation factors (IF-1, IF-2, and IF-3) aid in the formation of the 30S initiation complex. The antibiotic *streptomycin* (which was discovered in Rutgers) binds to the 30S subunit and distorts its structure, interfering with the initiation of protein synthesis. (2) GTP is then cleaved and initiation factors are released when the 50S subunit arrives to from the 70S initiation complex. (3) Then, there are some prokaryotic elongation factors: EF-Tu and Ef-Ts that direct binding of the appropriate tRNA to the codon in the empty-A site. *Tetracyclines* can act at this juncture and interact with small ribosomal subunits, blocking access of the aminoacyl-tRNA to the mRNA-ribosome complex. (4) Next, *peptidyltransferase*, an activity of the rRNA of the 50S ribosomal subunit, catalyzes peptide bond formation, transferring the initiating amino acid (or peptide chain) from the P site onto the amino acid at the A site. The antibiotic *chloramphenicol* inhibits prokaryotic peptidyltransferase and high levels may also inhibit mitochondrial protein synthesis. *Puromycin*can also act here and bears a structural resemblance to aminoacyl-tRNA. It becomes incorporated into the growing peptide chain, thus causing inhibition of further elongation in both prokaryotes and eukaryotes. It gets attached, but can't be extended. This can't be used to treat bacterial infection but has been used as protein synthesis inhibitor in the lab. (5) Next, the ribosome moves a distance of three nucleotides along the mRNA in the 5'→3' direction. What was in the P site is now in E; what was in the A site is now in P, and A is empty. GTP on EF-G is hydrolyzed. *Clindamycin* and *erythromycin* can act here and bind irreversibly to a site on the 50S subunit of the bacterial ribosome, thus inhibiting translocation. *Diphtheria toxin* can also act here to inactivate the *eukaryotic* elongation factor, EF-2, thus preventing translocation. (6) After this, Steps 3, 4, and 5 are repeated until a termination codon is encountered at the A site. (7) Lastly, a termination codon is recognized by a release factor (RF), which results in release of the newly synthesized protein. The synthesizing complex dissociates. GTP on RF-3 is hydrolyzed.

How does acetylation modification differ from methylation?

*Acetylation* is a characteristic of active *euchromatin*, but with *methylation* is variable. It depends where you're methylating as to whether that will increase of decrease gene transcription.

What are the eukaryotic RNA polymerases?

*Class → Products [Location]* *RNA pol I* → rRNA [Nucleolus] *RNA pol II* → HnRNA (mRNA) [Nucleoplasm] *RNA pol III* → tRNA and 5S RNA [Nucleoplasm]

What are the major inhibitors of protein synthesis and their modes of action?

*Inhibitor → Mode of Action* 1. *Streptomycin*: Prevents binding at the P site of the 30S subunit and inhibits initiation. It also causes misreading of the mRNA. 2. *Tetracycline*: Inhibits binding of aminoacyl tRNAs to the A site of the 30S subunit. Inhibits elongation. 3. *Chloramphenicol*: Inhibits peptidyl transferase activity of 50S subunit. 4. *Erythromycin*: Binds to 50S subunit and inhibits translocation. 5. *Puromycin*: Structural analog of aminoacyl tRNA. Reacts with peptidyl tRNA to give puromycin-peptidyl tRNA 6. *Cycloheximide*: Inhibits peptidyl transferase activity of 60S subunit (eukaryotic inhibitor)

(1) What is mRNA?

*Messenger RNA (mRNA)* molecules are the transcripts of DNA sequences that direct the *synthesis of proteins* (with assistance from rRNA and tRNA). They vary in chain length from a few hundred to several thousand nucleotides. The genetic material of some RNA viruses represents a special class of mRNA which can serve as a template for its own replication by an RNA-dependent RNA polymerase.

What are microRNAs (miRNAs)?

*Micro RNAs (miRNAs)* are transcribed from DNA but do not code for proteins. They regulate gene expression by effects on either mRNA or on translation that depend on complementarity to mRNA.

(4) What are noncoding RNAs?

*Noncoding RNAs* are small RNAs which include snRNAs, miRNAs, siRNAs, and piRNAs. *Small nuclear RNAs (snRNAs)* are involved in *splicing of mRNAs*. Regulatory RNAs include the *micro RNAs (miRNAs)* and *short interfering RNAs (siRNAs)*. Another type of small RNA, *piRNAs*, control gene expression in germline cells. *Long non-coding RNAs* (long ncRNAs, lncRNA) are in general considered as *non-protein coding transcripts* longer than 200 nucleotides. Some have been found to have a role in the regulation of gene transcription and translation.

What are P-bodies?

*P-bodies* are sites for RNA degradation and storage. The term P-bodies has been given to cytoplasmic particles where mRNA is stored and degraded. Degradation of mRNA requires the action of a decapping enzyme in the P-bodies that removes the methylated guanosine at the 5' end. Micro-RNAs (miRNAs) regulate mRNA translation and mRNAs subject to miRNA repression accumulate in P-bodies.

What is the role of Class I eukaryotic RNA polymerase?

*RNA polymerase I* is responsible for making most of the ribosomal RNA (rRNA). It makes the 28S and 18S and 5.8S, but not the 5S RNA (which is made by the *RNA polymerase III*). Therefore, three of the four eukaryotic ribosomal RNAs are made by RNA polymerase I

What is the role of Class III eukaroytic RNA polymerase?

*RNA polymerase III* makes the *transfer RNA* (tRNA) species and the *5S rRNA* and one or two other RNAs.

(2) What is rRNA?

*Ribosomal RNA (rRNA)* is a structural component of ribosomes.

(3) What is tRNA?

*Transfer RNA (tRNA)* molecules are the adapters or carriers of amino acids for protein synthesis. The tRNA gets covalently bound to an amino acid via an anticodon sequence that is complementary to the codon sequence on mRNA. All have a characteristic *clover leaf secondary structure*. In addition to the usual bases of adenine, guanine, uracil and cytosine, tRNAs have many rare bases, such as *7-methylguanine* and *pseudouridine*. All tRNA molecules terminate at the 3' end in the sequence pCpCpA (CCA).

What are the stop codons?

*UGA, UAA and UAG* U Go Away, U Are Away, U Are Gone.

What are some inhibitors of RNA synthesis?

1. *Enzyme specific inhibitors*: a. *Rifampicin (Rifamycin)*: inhibitor of E. coli RNA polymerase; clinical use for tuberculosis [inhibits initiation of RNA synthesis] b. *Alpha-amanitin*: has no clinical use but RNA polymerase II is most sensitive to this toxin so it can be used as a tool to measure if RNA polymerase II is involved in a particular process. [RNA pol II synthesizes hnRNA (mRNA)] 2. *DNA intercalating agents*: (less specificity) a. *Actinomycin D*: has some use as a cancer chemotherapeutic agent [inhibits elongation of RNA sythesis] b. *Quinacrine*

What are the four steps in protein synthesis that involve the hydrolysis of high energy phosphate bonds?

1. ATP is converted to AMP and PPi in the formation of the aminoacyl-tRNA. (Figure 6-56) 2. ATP is hydrolyzed in scanning and GTP is hydrolyzed to GDP and Pi in the binding of an aminoacyl-tRNA to the A site. (Figure 6-71 and Figure 31.13, step 3) 3. GTP is hydrolyzed to GDP and Pi in the translocation from the A site to the P site. (Figure 31.13, step 5) 4. GTP is hydrolyzed to GDP and Pi in the binding of fmet-tRNA to the P site (Figure 31.13, step 2). This step occurs only once in the synthesis of a protein but the regeneration of the other nucleotides requires four high energy phosphate bonds for each peptide bond that is synthesized.

What is the lecture outline?

1. Codons 2. Activation of amino acids 3. Initiation of peptide synthesis 4. Elongation 5. Termination 6. Energy requirements 7. Signal sequences and location of proteins 8 Comparison of eukaryotes and prokaryotes 9. Inhibitors

The genetic information in DNA is transcribed into what four classes of RNA?

1. Messenger RNA (mRNA) 2. Ribosomal RNA (rRNA) 3. Transfer RNA (tRNA) 4. Noncoding RNAs

What are the various post-transcriptional modifications of some amino acid residues?

1. Phosphorylation 2. Glycosylation 3. Hydroxylation 3. Carboxylation 4. Biotinylated enzyme 5. Farnesylated enzyme

What is required for binding of the RNA polymerase to occur?

Binding of the enzyme requires localized and transient *opening of the DNA duplex*.

What are the other transcriptional activator proteins that play a role in initiation of transcription? (Figure 6-19)

Activator proteins such as enhancers and binding of general transcription factors, RNA polymerase, mediator, chromatin remodeling complexes, and histone acetylases all play a role and dictate not only where the RNA polymerase will start transcription but how active it is.

(4) Describe *carboxylation* as a post-transcriptional modification.

Additional carboxyl groups can be added to glutamate residues by vitamin K-dependent carboxylation. The resulting γ-carboxyglutamate (Gla) residues are essential for the activity of several of the blood-clotting proteins. This carboxylation provides a system good for calcium binding. Remember that various protein cloting factors such as prothrombin and Factor 7, 9, and 10 are calcium-binding proteins that therefore require this modification.

What is unique about the toxin alpha-amanitin?

Alpha amanitin is a mushroom toxin that shuts down liver function. *RNA polymerase II* is distinguished by its sensitivity to inhibition by the toxin. This means that we can selectively inhibit RNA polymerase Class II and not Class I or Class III at certain concentrations.

How can we end up with more proteins than we have genes encoding for them?

Alternative splicing and alternative sites for poly-adenylation contirbute to this phenomenon that while we may only have 25,000 genes, these variabilites in how we modify RNA has consequences for the number of proteins that can be formed. We thus end up making many more proteins than just 25,000 different proteins.

Describe the schematic illustration of an "export-ready" mRNA molecule and its transport through the nuclear pore. (Figure 6-40)

As indicated, some proteins travel with the mRNA as it moves through the pore, whereas others remain in the nucleus. Once in the cytoplasm, the mRNA continues to shed previously bound proteins and acquire new ones; these substitutions affect the subsequent translation of the message. Because some are transported with the RNA, the proteins that become bound to an mRNA in the nucleus can influence its subsequent stability and translation in the cytosol. RNA export factors, shown in the nucleus, play an active role in transporting the mRNA to the cytosol. Some are deposited at exon-exon boundaries as splicing is completed, thus signifying those regions of the RNA that have been properly spliced. We have helpers throughout this process including the CBC (cap binding complex), Ser/Arg rich proteins , hnRNP proteins, poly-A binding proteins, nuclear pore complex, and eukaryotic initiation factors for protein synthesis.

(6) Describe *farnesylated enzyme* as a post-transcriptional modification.

Attachment of lipids, such as farnesyl groups, can help *anchor proteins to membranes*. Farnesyl groups are added through the cysteine residue, which *adds a big hydropobic group*. RAS protein gets modified by this modification. The large hydrophobic unit is good for binding of some proteins to the cell membrane.

(5) Describe *biotinylated enzyme* as a post-transcriptional modification.

Biotin is covalently bound to the ε-amino groups of lysine residues of biotin-dependent enzymes that catalyze carboxylation reactions, such as *pyruvate carboxylase* (in gluconeogenesis) and *acetyl-CoA carboxylase* (in fatty acid synthesis).

What are the properties of RNA and DNA polymerases?

Both RNA and DNA Polymerases require a DNA template and four nucleoside triphosphates for activity. Nucleic acid synthesis requires *base pairing* and proceeds in *5' to 3' direction*, antiparallel to the template strand. *RNA polymerases, unlike DNA polymerase, don't require a primer*. They require a template, but not a primer! RNA polymerase is able to use a duplex template to start a chain and to terminate synthesis before the end of the template. There is also *no proofreading or repair in RNA synthesis*, so the fidelity of the transcription can be less than that of DNA replication. Furthermore, *only one strand of the DNA duplex is transcribed in RNA synthesis*. However, on any DNA molecule, all the transcribed sequences for individual genes need not be on the same DNA strand.

What are the consequences of altering the nucleotide sequence of a codon?

Changing a single nucleotide base on the mRNA chain (a "point mutation") can lead to any one of three results: 1. Silent mutation 2. Missense mutation 3. Nonsense mutation

Summarize the flow of genetic information. [Figure 31.16]

DNA is an information molecule that codes sequence of deoxyribonucleotides. DNA provides information transfer by *transcription* resulting in synthesis of *mRNA*, which is an information molecule, coding by sequence of ribonucleotides. mRNA provides information transfer by *triplet codons* defined by the genetic code and characterized as specific, universal, redundant, nonoverlapping, and commaless. The triplet codons pair with specific *anticodons* contained in *aminoacyl-tRNAs*, which are synthesized by a specific tRNA that recognizes aminoacyl-tRNA synthetase which recognizes a specific amino acid. Aminoacyl-tRNAs deliver amino acids to *ribosomes* resulting in synthesis of *protein*, which is a functional molecule, consisting of sequence of amino acids.

Summarize the flow of genetic information. [Lecture Notes]

DNA is transcribed to give RNAs, which includes mRNA. The basis for protein synthesis is then the triplet codon, starting with methionine (eukaryotes) or formyl-methionine (prokaryotes) and adding on to that. Base complementarity is needed between the anticodon on tRNA and the codon on mRNA. The clover-leaf structure of tRNA is distinctive, but its true structure is more complicated. Note that internal base-pairing is a big feature of tRNA structure. Ribosomes are made of a limited number of ribosomal RNAs, but also a large number of protein comprised of large and small subunits. The total is 70S in prokaryotes and 80S in eukaryotes. Finally, we have synthesis of the protein starting from the N-terminal moving towards the C-terminal.

Central dogma: ______ to ______ to ___________.

DNA to RNA to protein.

Describe elongation in Figure 6-65.

Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. Because only one of the many types of tRNA molecules in a cell can base-pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. The three-step cycle shown is repeated over and over during the synthesis of a protein. An aminoacyl-tRNA molecule binds to a vacant A-site on the ribosome in *step 1*, a new peptide bond is formed in *step 2*, and the mRNA moves a distance of three nucleotides through the small-subunit chain in *step 3*, ejecting the spent tRNA molecule and "resetting" the ribosome so that the next aminoacyl-tRNA molecule can bind. Although the figure shows a large movement of the small ribosome subunit relative to the large subunit, the conformational changes that actually take place in the ribosome during translation are more subtle. It is likely that they involve a series of small rearrangements within each subunit as well as several small shifts between the two subunits. 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. The position at which the *growing peptide chain is attached to* a tRNA does not change during the elongation cycle: it is always linked to the tRNA present in the *P site* of the large subunit.

Describe termination of peptide synthesis.

Elongation continues until a stop codon is reached. This is a unique case in which *prokaryotes have more termination factors than eukaryotes*. Prokaryotes have termination factors (RF-1, RF-2 and RF-3) that facilitate the release of the polypeptide. In eukaryotes, there is a single release factor eRF.

Which drug blocks elongation in RNA synthesis?

Elongation is blocked by the antibiotic, *actinomycin D*, which binds to and intercalates between G-C base pairs. It has some use as a cancer chemotherapeutic agent.

Where is the CCA sequence derived from in prokaryotes and eukaryotes?

In *prokaryotes*, the 3' end CCA sequence of all tRNA molecules is derived from the original transcript of the tRNA. In *eukaroytes*, the CCA sequence is added afterwards to the modified transcript.

What are the roles of snRNAs?

In association with proteins, uracil-rich *small nuclear RNAs (snRNA)* form *small nuclear ribonucleoprotein particles (snRNPs*, or "snurps" designated as U1, U2, etc.) that *mediate splicing*. They facilitate the *removal of introns* by forming base pairs with the consensus sequences at each end of the intron.

Summarize eukaryotic transcription: DNA-directed RNA synthesis.

Eukaryotic transcription (DNA-directed RNA synthesis) consists of *initiation, elongation, termination, and posttranscriptional modification*. *Initiation* requires binding of protein and transcription factors and RNA polymerase to promoter sites at the beginning of a gene, which is facilitated by enhancer-binding transcription factors bound to enhancer sequences at sites far from the gene. *Elongation* requires local unwinding of the DNA helix by RNA polymerase, followed by synthesis of 5'→3' RNA transcript coded for by the DNA template read in the 3'→5' direction. *Termination* requires a termination signal sequence and results in release of RNA polymerase and newly synthesized transcript from DNA. *Posttranscriptional modifications* include splicing of mRNA exons to eliminate non-coding introns, cleavage and trimming of pre-ribosomal RNAs, trimming and base modification in tRNA, addition of a 3'-poly-A "tail" and a 5'-7-methylguanosine "cap" to mRNA.

How does base pairing with target RNAs distinguish miRNA and siRNA?

For control of expression of mRNA there has to be some base complementarity, but there are two types of control. siRNA works by binding to target RNA and results in degradation of that RNA, i.e. *RNA cleavage*. miRNA doesn't require strict base complementarity with the target RNA, but results in *inhibition of translation* (and thus protein synthesis). miRNAs are naturally occurring regulatory mechanisms and siRNAs have applications in experimental chemistry and are used in therapeutics for regulation of specific gene expression.

Describe the process of removal of introns and splicing of RNA.

Four different splicing mechanisms have been found. In the most common mechanism, the splicing is catalyzed by specialized RNA-protein complexes called *small nuclear ribonucleoprotein particles (snRNPs)*. The RNAs found in snRNPs are identified as *U1, U2, U4, U5 and U6*. The genes encoding these snRNAs are *highly conserved* in evolution. Analysis of a large number of mRNA genes has led to the identification of highly conserved consensus sequences at the 5' and 3' ends of essentially all mRNA introns. The *U1 RNA* has sequences that are complimentary to sequences near the 5' end of the intron. The binding of U1 RNA distinguishes the GU *at the 5' end* of the intron from other randomly placed GU sequences in mRNAs. The *U2 RNA* also recognizes sequences in the intron, in this case near the *3' end*. The addition of U4, U5 and U6 RNAs forms a complex identified as the *spliceosome* that then removes the intron and joins the two exons together.

What are the four types of processing of RNA transcripts?

Four types of processing: 1. Endonucleolytic cleavage 2. Terminal additions (ex. 5' guanine cap and 3' poly A tail on eukaryotic mRNAs) 3. Nucleoside modification (post-transcriptional) 4. Splicing

What is I-cell disease?

In *I-cell disease* (mucolipidosis II), there is misdirection of several proteins because they contain a mannose residue instead of *mannose 6-phosphate* which directs proteins to the lysosomes.

(1) Describe binding of RNA polymerase in RNA synthesis.

In *prokaryotes*, *RNA polymerase* binds to DNA at specific sites called *promoters* (Pribnow box - TATAAT sequence) near the beginning of each transcription unit. The sigma unit must be present to prevent nonspecific binding at other sites on the DNA. Note that in *eukaryotes*, some additional sequences called *enhancer elements* also direct RNA polymerase to specific promoters.

How do nucleotide sequences in mRNA signal where to start protein synthesis?

In eukaryotes, the initiator tRNA (which is coupled to methionine) is first loaded into the small ribosomal subunit along with additional proteins called eucaryotic initiation factors, or eIFs (Figure 6-71). Of all the aminoacyl tRNAs in the cell, only the methionine-charged initiator tRNA is capable of tightly binding the small ribosome subunit without the complete ribosome present. Next, the small ribosomal subunit binds to the 5' end of an mRNA molecule, which is recognized by virtue of its 5' cap and its two bound initiation factors, eIF4E (which directly binds the cap) and eIF4G. The small ribosomal subunit then moves forward (5' to 3') along the mRNA, searching for the first AUG. This movement is facilitated by additional initiation factors that act as ATP-powered helicases, allowing the small subunit to scan through RNA secondary structure. In 90% of mRNAs, translation begins at the first AUG encountered by the small subunit. At this point, the initiation factors dissociate from the small ribosomal subunit to make way for the large ribosomal subunit to assemble with it and complete the ribosome. The initiator tRNA is now bound to the P-site, leaving the A-site vacant. Protein synthesis is therefore ready to begin with the addition of the next aminoacyl tRNA molecule.

In eukaryotes, the scanning process requires ______, and elongation phase requires ______.

In eukaryotes, the scanning process requires *ATP*, and elongation phase requires *GTP*.

Summary of the steps leading from gene to protein in eukaryotes.

In eukaryotic cells, the RNA molecule (i.e. primary transcript) produced by transcription alone would contain both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm. Although these steps are depicted as occurring one at a time, in a sequence, in reality they are coupled and different steps can occur simultaneously. For example, the RNA cap is added and splicing typically begins before transcription has been completed. Because of this coupling, complete primary RNA transcripts do not typically exist in the cell.

Summary of the steps leading from gene to protein in prokaryotes.

In prokaryotes, the production of mRNA molecules is much simpler. Note that *prokaryotes don't have intron sequences*. 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. Since prokaryotic cells lack a nucleus, transcription and translation take place in a common compartment. In fact, translation of a bacterial mRNA often begins before its synthesis has been completed.

Compare heterochromatin and euchromatin.

In the nucleus, not all the genes are being expressed. *Heterochromatin* is comprised of *condensed chromatin*, associated with low expression of gene transcription. *Euchromatin*, on the other hand, is the *more extended and open chromatin*, which is actively transcribed. Factors distinguishing the two can be the *side chain modifications* of histones. For example, at the H3 histone, there is *variable methylation*. Methylation at the *9 and 27 position* is associated with inactivation and thus *heterchromatin*, whereas methylation of lysine at the *4 position* is associated with *euchromatin*.

What are codons?

In the translation of the information in mRNA to produce a protein, the sequence of amino acids is determined by *triplet sequences of bases* in the mRNA, called a *codon*. An amino acid may be specified by more than one codon since there are *64 possible codons* of the four bases and there are codons for only *20 amino acids*. There are three codons that are *stop codons* and which terminate transcription: *UGA, UAA, and UAG*.

Where is the aminoacyl group added on the tRNA?

Looking at the tRNA, the 3' end has CCA and it is to this 3'-OH that you add the aminoacyl group.

(2) Describe *glycosylation* as a post-transcriptional modification.

Many of the proteins that are destined to become part of a plasma membrane or to be secreted from the cell, have carbohydrate chains attached to the amide nitrogen of asparagine (N-linked) or the hydroxyl groups of serine, threonine, or hydroxylysine (O-linked). *N-glycosylation* occurs in the *endoplasmic reticulum* and *O-glycosyation* in the *Golgi*. Glycosylation is also used to target proteins to the matrix of lysosomes. Lysosomal acid hydrolases are modified by the phosphorylation of mannose residues at carbon 6.

What is a major transcription factor on the TFIID complex?

One of the major proteins in this complex TFIID is a *TATA binding protein (TBP)*, which facilitates transcription, binds to the promoter site, and helps in localizing RNA polymerase, because RNA pol II by itself doesn't know where to start, but with association of certain factors you build a complex.

Summarize RNA structure.

RNA structure consists of ribose, phosphate in a diester linkage, and bases (adenine, uracil, cytosine, and guanine), which produce ribonucleotides. Polymers of these ribonucleotides form rRNA, tRNA, and mRNA. *rRNA* functionis as the structural component of ribosomes. Structural features include: association with protein, three size species in prokaryotes (5S, 23S, and 16S), and four size species in eukaryotes (5S, 28S, 5.8S, and 18S). *tRNA* functions as an adaptor molecule that carries a specific amino acid to the ribosome/mRNA complex. Structural features include: unusual bases, extensive intra-chain base pairing, having at least one specific type of molecule for each of the 20 amino acids found in proteins, and the 3'-CCA. *Eukaryotic mRNA* functions as the template for protein synthesis. Structural features include: 3'-Poly-A "tail" and 5'-cap of 7-methylguanosine.

Describe the initiation phase of protein synthesis in eukaryotes. (Figure 6-71).

Only three of the many translation initiation factors required for this process are shown. Efficient translation initiation also requires the poly-A tail of the mRNA bound by poly-A-binding proteins which, in turn, interact with eIF4G. In this way, the translation apparatus ascertains that both ends of the mRNA are intact before initiating. Although only one GTP hydrolysis event is shown in the figure, a second is known to occur just before the large and small ribosomal subunits join. Our first aminoacyl tRNA is a methioninyl tRNA. We know this is a eukaryotic system because of the poly-A tail on the 3' end, the 5' cap, and the eIF. In the scanning process, whereby the small subunit of ribosome moves along till it finds an AUG. That process of scanning for an AUG site requires ATP. The anticodon on tRNA will base pair with AUG and will bring large ribosomal subunit. The GTP that was involved is converted to GDP. The second aminoacyl-tRNA with amino acid "aa" comes in at A site. Now we have formation of first peptide bond between methionine and the second amino acid. tRNA movement ends up on E site and the tRNA which was at A site will be on P site now. Eventually, the empty tRNA falls off the E site. So now we have an empty A site where the 3rd amino acid comes in.

(1) Describe *phosphorylation* as a post-transcriptional modification.

Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. This phosphorylation is catalyzed by one of a family of *protein kinases* and may be reversed by the action of cellular protein *phosphatases*. The phosphorylation may increase or decrease the functional activity of the protein. Several examples of these phosphorylation reactions have been previously discussed, for example, for the regulation of synthesis and degradation of glycogen. Phosphorylation remember is an inhibitor of glycogen synthase and an activator of glycogen phosphorylase.

Why is transcription in prokaryotes more rapid than in eukaryotes?

Prokaryotes have an advantage in that they don't have compartmentation of the various functions. In eukaryotes, DNA an RNA synthesis is occurring in the nucleus and translation in the cytoplasm. In prokaryotes, protein synthesis can begin even before mRNA synthesis is finished. The process is thus more rapid in prokaryotes.

(3) Describe *hydroxylation* as a post-transcriptional modification.

Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C-dependent hydroxylases in the endoplasmic reticulum. Remember, that with scurvy, this process is deficient because it requires vitamin C.

How do signal sequence guide the location of proteins?

Protein synthesis occurs in the cytoplasm but many proteins become associated with specific cellular organelles or are secreted. - Proteins to be located in the *cytosol, mitochondria or nuclei* are synthesized on polyribosomes free in the cytosol. - Proteins that end up in the nucleus, such as histones, need a nuclear localization signal (lysine and arginine). - Proteins to be located in *cellular membranes, the lysosomes or for extracellular transport* are synthesized in association with the rough endoplasmic reticulum and the Golgi complex. Synthesis for membrane bound protein begins with an *N-terminal hydrophobic signal sequence*. Proteins may then be transported to the Golgi complex in vesicles which bud off the rough endoplasmic reticulum. The Golgi serves as a sorting mechanism for the directing of protein transport. Additionally, the location of a protein may be influenced by a signal peptidase which cleaves the signal sequence.

What are the major differences between DNA and RNA?

RNA has *ribose*, uses *uracil*, is usually *single stranded*, and RNA molecules are smaller. DNA has *deoxyribose*, uses *thymine*, is usually *double-stranded*, and DNA molecules are bigger.

(2) Describe initiation of RNA synthesis.

RNA polymerase has a specific binding site for the initiating triphosphate (ATP or GTP) and another for triphosphates added during elongation.

Where does ribosomal RNA synthesis take place within the cell?

Ribosomal RNA (rRNA) synthesis is associated with the *nucleolus* in eukaryotic cells.

Describe the composition of ribosomes.

Ribosomes are large complexes of protein and ribosomal RNA. They consist of two subunits—one large and one small—whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values. Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive.

What is the Svedberg unit?

S represents the *Svedberg unit*, a measure of the *rate of sedimentation* when a molecule is subjected to a centrifugal force. The Svedberg unit is a measure of both the *molecular weight* of a molecule and its *conformation*.

Describe processing of intervening sequences and splicing.

Sequencing of bases in DNA of eukaryotic cells has revealed that, unlike prokaryotic cells, there are segments of the transcribed RNA that are not present in the final RNA product used in protein synthesis. It is *necessary to remove these intervening sequences*, which have been called *introns*, and then splice the sequences called *exons* to form the mRNA molecule. Genes have been identified with many introns or none. The function of introns is not clear but it has been suggested that the existence of exons facilitates reorganization of protein domains in evolution. *Splicing* involves specific splice sites and the formation of *lariat structures* as an intermediate. There are specific sequences associated with splice sites and that is how factors involved in splicing have recognition of the appropriate splice sites.

What are siRNA?

Small (or short) interfering RNA (siRNA) is a class of double-stranded RNA molecules, 20-25 base pairs in length, similar to miRNA, and operates within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription. It is the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein coding genes. siRNA is a synthetic RNA duplex designed to specifically target a particular mRNA for degradation.

How do RNA viruses make DNA?

Some viruses use *reverse transcriptase* where they use RNA template to make DNA. There are also other RNA viruses that use RNA to make RNA.

What are the steps in synthesis of RNA?

Synthesis of RNA requires the following steps: 1. Binding 2. Initiation 3. Elongation 4. Termination

What are the consensus sequences in the eukaryotic promoter region?

The *eukaryotic promoter* contains consensus sequences: the *TATA* (or Hogness box) and the *CAAT box*.

What are the proteins and RNA species that comprise the prokaryotic and eukaryotic ribosomes?

The *prokaryotic 70S ribosome* is comprised of the *50S* subunit (comprised of 5S RNA, 23S RNA, and 32 proteins) and the *30S* subunit (comprised of 16S RNA and 21 proteins). The *eukaryotic 80S ribosome* is comprised of the *60S* subunit (comprised of 5S RNA, 28S RNA, 5.8S RNA, and 50 proteins) and the *40S* subunit (comprised of 18S RNA and 30 proteins).

What are the consensus sequences in the prokaryotic promoter region?

The *prokaryotic promoter* contains characteristic consensus sequences including *-35 sequence* and *Pribnow box*

What is the Pribnow box?

The Pribnow box is the sequence *TATAAT* that is an essential part of a promoter site on DNA for transcription to occur in *prokaryotes*.

How is DNA transcribed by the enzyme RNA polymerase? (Figure 6.8) [read fast - he skipped this slide]

The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here, small "T" shapes) one by one to the RNA chain at the polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single-stranded complementary copy of one of the two DNA strands. The polymerase has a rudder that displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only transiently, and a "window" of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate-phosphate bonds provides the driving force for the polymerization reaction.

What are the subunits of the RNA polymerase of E.coli?

The RNA polymerase of E. coli consists of 5 subunits (β,β', 2α and ω subunits) together with a dissociable sigma subunit.

What is the TATA box?

The TATA box is a DNA sequence found in the *promoter region* of genes in *eukaryotes*. It is considered to be the core promoter sequence and it is the binding site of either general transcription factors or histones and is involved in the process of transcription by RNA polymerase.

Describe activation of amino acids.

The activation of amino acids for protein synthesis involves the formation of aminoacyl-tRNA molecules. The activation is catalyzed by specific *aminoacyl tRNA synthetases* and occurs in 2 steps with the formation of an intermediate aminoacyl-adenylate: (1) amino acid + ATP → aminoacyl-adenylate + pyrophosphate (2) aminoacyladenylate + tRNA → aminoacyl-tRNA + AMP This process requires ATP and the aminoacyl tRNA synthetase first makes aminoacyl-adenylate, with release of pyrophosphate (pyrophosphatase activity then converts it to 2 molecules of orthophosphate). From aminoacyladenylate, the aminoacyl group is transferred to tRNA and it is in the form of aminoacyl-tRNA that we get incorporation of the amino acid in to the growing amino acid chain.

Describe elongation in peptide synthesis.

The aminoacyl tRNA recognized by the codon next to the AUG sequence binds to the A site. In bacteria there are three elongation factors (EF-Tu, EF-Ts and EF-G). In eukaryotes, the factors are EF1a, EF1b and EF2. A *peptidyl transferase* catalyzes the formation of a peptide bond between the formyl methionine or methionine and the second amino acid. This is one case where you have an enzyme that is an RNA and not a protein. The overall process results in a naked tRNA at the P site and a dipeptide attached to tRNA at the A site. The dipeptidyl tRNA displaces the naked tRNA at the P site leaving a vacant A site for the incoming aminoacyl tRNA bearing the third amino acid. The formation of peptide bonds continues with the sequential addition of amino acids. The elongation process involves the hydrolysis of GTP. mRNA is translated in a 5' to 3' direction. The protein is synthesized from the N to the C terminal.

Why is it interesting that mitochondria has sensitivity to the antibiotic chloramphenicol?

The antibiotic *chloramphenicol* has use in inhibiting bacterial protein synthesis, but also has some action on mitochondrial protein synthesis. Remember, mitochondria evolutionarily had prokaryotic origin, so it makes sense that they retained sensitivity to this antibiotic.

What is the mechanism of splicing? (Figure 30.18)

The binding of snRNPs brings the sequences of the neighboring exons into the correct alignment for splicing. The 2'-OH group of an adenosine (A) residue (known as the branch site) in the intron attacks the phosphate at the 5'-end of the intron (splice donor site), forming an unusual 2'→5' phosphodiester bond and creating a *"lariat" structure*. The newly freed 3'-OH of exon 1 attacks the 5'-phosphate at the splice acceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded. After introns have been removed and exons joined, the mature mRNA molecules leave the nucleus and pass into the cytosol through pores in the nuclear membrane.

What is the central dogma theory? (Fig. 6.2)

The central dogma states that DNA is replicated or transcribed to give RNA and RNA is then translated to give proteins.

Which drug block initiation of RNA synthesis?

The formation of the first bond in initiation of RNA synthesis is blocked by the antibiotic, *rifampicin* (rifamycin, rifampin). Rifampicin is drug used in treatment of tuberculosis. It is an inhibitor of prokaryotic RNA synthesis, but not of eukaryotic RNA synthesis.

What are the differences in rRNA between prokaryotes and eukaroytes?

There are *three* discrete size classes of rRNA in *prokaryotes* and *four* in *eukaryotes*. These are usually referred to by their sedimentation coefficients as *5S, 16S and 23S* in prokaryotes, and *5S, 5.8S, 18S and 28S* in eukaryotes.

Describe the genetic code table to translate the codon.

The genetic code table is a way that the triplet codons are written. Note that some amino acids have multiple codons; for example, there are 6 different codon sequences for leucine, whereas some are unique (ex. methionine and tryptophan). There can therefore be anything between 1 and 6 codons for a particular amino acid. The codon 5'-AUG-3' codes for methionine (see Figure 31.2). Note AUG is the initiation (start) codon for translation. Sixty-one of the 64 codons code for the 20 common amino acids (remember three are the stop codons: UGA, UAA, UAG).

Describe initiation of peptide synthesis.

The initiation of polypeptide synthesis occurs with the binding of *methionine tRNA* to an AUG sequence on the messenger RNA. The codon is recognized by the anticodon of tRNA and not by the amino acid. The mRNA must be in association with the ribosomal subunits and initiation factors. GTP and magnesium ions are required. In bacteria the initiator tRNA carries *N-formyl methionine* (fmet-tRNA). In *prokaryotes*, there are 3 proteins that serve as initiation factors (IF-1, IF-2 and IF-3). In *eukaryotes* there are at least 10 initiation factors (eIF-1 etc.). There are two important sites in the initiation complex known as the *peptide site (P)* and the *acceptor site (A)*. The *initiator aminoacyl tRNA binds to the P site*, and then every amino acid added after that will come in at the *A site* on the ribosome.

What are the modifications that occur at the two ends of the RNA transcript in eukaryotes?

The modification of the two ends is something you don't see in prokaryotes. At the *5' end*, there is addition of a *5' cap* (guanine with 3 phosphates). At the *3' end*, there is addition of a *poly-A sequence* that is not coded by the original gene. Both of these modification give *stability* to the mRNA. Once you've had these modifications, the mRNA can be exported out to cytoplasm for translation.

The prokaryotic ______ and ______ ribosomal subunits together form a ______ ribosome. The eukaryotic ______ and ______ subunits form an ______ ribosome.

The prokaryotic *50S* and *30S* ribosomal subunits together form a *70S* ribosome. The eukaryotic *60S* and *40S* subunits form an *80S* ribosome.

Describe processing of transfer RNA (tRNA).

The tRNA molecules are formed from large precursors in all cells. Processing involves *nucleolytic cleavage, nucleotide modifications and folding* to generate a partial double-stranded character. The *CCA* terminus at the *3' end* may be coded in the gene as in E. coli or it may be added post-transcriptionally as in eukaryotes (in mammalian cells, the CCA addition is a cytoplasmic function).

The template strand is the ________________ strand and the nontemplate strand is the ________________ strand.

The template strand is the *antisense* strand and the nontemplate strand is the *sense* strand.

(4) Describe termination of RNA synthesis.

The termination sequences on DNA (forming base paired *hairpin structures* in RNA) act to signal termination of RNA synthesis. These sequences are recognized by the RNA polymerase and the following dissociation of the RNA transcript is facilitated by a protein called the *rho factor*.

Describe amino activation in Figure 6-56.

The two-step process in which an amino acid (with its side chain denoted by R) is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The *amino acid* is first activated through the linkage of its carboxyl group directly to an AMP moiety, forming an *adenylated amino acid*. The linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the *AMP-linked carboxyl group* on the amino acid is then transferred to a *hydroxyl group* on the sugar at the *3' end of the tRNA molecule*. This transfer joins the amino acid by an activated *ester linkage* to the tRNA and forms the *final aminoacyl-tRNA molecule*.

How can eukaryotic and prokaryotic translation mechanisms be specifically inhibited?

There are many differences between the translation mechanism in eukaryotes and prokaryotes which make possible specific inhibition of bacterial or mammalian protein synthesis. There are selective for prokaryotic protein synthesis (streptomycin, chloramphenicol, tetracycline and erythromycin) and for eukaryotic protein synthesis (cycloheximide). *Puromycin, however, affects protein synthesis in all systems*.

Compare the promoters in prokaryotes and eukaryotes.

There are upstream promoters in prokaryotes, whereas eukaryotes also have upstream or downstream enhancer sequences. *Eukaryotes* contain the *TATA box*, while *prokaryotes* contain something called the *Pribnow box* (TATAAT). Not all eukaryotic genes have the TATA box and are thus dependent on further upstream sequences and on other promoter sequences which may be downstream of the start site.

Describe processing of ribosomal RNA (rRNA).

There is an extensive cleavage of ribosomal precursor RNA in all cells. In eukaryotes, the 80S ribosomal particle has two subunits (40S and 60S). The 40S subunit has one RNA molecule (18S) and the 60S subunit has three RNA molecules (5S, 5.8S and 28S).

Describe processing of heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA).

There is much less processing of prokaryotic mRNA than of eukaryotic mRNA. In mammalian cells, *mRNA* is derived from large precursor molecules called *heterogeneous nuclear RNA (HnRNA)* which are 10-100 times larger than RNA found in the cytoplasm. The initial transcript is much longer than the final mRNA, in that *introns* have to be *spliced out*. In addition to *nucleolytic cleavage*, the processing of HnRNA involves the *addition of 7-methylguanosine* at the 5'-terminal. This process results in the structure 7-methylGpppXp... and is known as *capping*. There is also *addition of poly(A) tails* to the 3' terminals of most but not all RNA species. The *signal sequence AAUAAA* indicates where to cut the RNA and then *add the poly-A tail*.

Describe the functions of the transcription factors involved in initiation of transcription.

To begin transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, and so on). The promoter contains a DNA sequence called the *TATA box*, which is located 25 nucleotides away from the site at which transcription is initiated. The *TATA box* is recognized and bound by transcription factor *TFIID* via *TATA binding protein (TBP)*. This enables the adjacent binding of *TFIIB*. TFIID essentially facilitates transcription, binds promoters at the appropriate site, and *helps localize RNA polymerase*. RNA polymerase II by itself doesn't know where to start, but with association of these factors, you start to build the complex. The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. *TFIIH* then uses ATP to pry apart the DNA double helix at the transcription start point, allowing transcription to begin.

How does DNA transcription produce a single-stranded RNA molecule that is complementary to one strand of DNA?

Transcription begins with opening 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 synthesis of an RNA molecule. This is the template strand (or *antisense strand*) and the other strand serves as the nontemplate strand (or *sense strand*), whose base sequence corresponds to base sequence of RNA transcript.

(2) What is a missense mutation?

With a *missense mutation*, the codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base—C—to become CCA, it will code for a different amino acid, in this case, proline. The substitution of an incorrect amino acid is called a "missense" mutation.

What are polysomes?

Translation begins at the 5'-end of the mRNA, with the ribosome proceeding along the RNA molecule. Because of the length of most mRNAs, more than one ribosome at a time can translate a message. This complex of one mRNA and a number of ribosomes is called a *polysome* or *polyribosome*. In this way, multiple proteins can be synthesized at the same time, by different ribosomes all on one mRNA molecule.

What happens with trinucleotide repeat expansion?

WIth *trinucleotide repeat expansion*, occasionally a sequence of three bases that is repeated in tandem will become amplified in number, so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the huntingtin protein, causing the neurodegenerative disorder, *Huntington disease*. The additional glutamines result in unstable proteins that cause the accumulation of protein aggregates. If the trinucleotide repeat expansion occurs in the untranslated regions of a gene, the result can be a decrease in the amount of protein produced as seen, for example, in *fragile X syndrome* and *myotonic dystrophy*. Note that in fragile X syndrome, the most common cause of intellectual disability, the expansion results in gene silencing through DNA hypermethylation.

Describe gene expression in eukaroytic cells. (Figure 6.90)

We have intervening sequences called *introns* that are not part of final mRNA, but are transcribed and then spliced out. One important mechanism in removing introns involved the *lariat structure*. We have modifications such as addition of 7-methylguanine with 3 phosphates at the 5' end called *5' capping* and the AAUAAA which indicated where to cut and at the *3' poly-A tail*. Once mRNA is formed and modified, it then gets out into the cytoplasm, becomes associated with the ribosome, and the information on mRNA gets read off in the formation of a protein. The proteins are synthesized starting at N terminal towards C terminal. After completion of protein synthesis and protein folding, then proteins eventually undergo turnover, i.e. protein degradation.

Explain the importance of tissue-specific RNA editing of apo-B pre-mRNA.

We know RNA is transcribed then spliced and capped and polyadenylated. However, RNA editing is a way of getting 2 different proteins from same gene and this pertains to the apoB protein. This mechanism works out differently in the liver and intestine, despite there being the same gene in both tissues. There is sequence UAA which is stop codon and another sequence upstream CAA which codes for amino acid. In the *liver*, there isn't any editing, so when this is used to make protein, the protein synthetic machinery puts in the appropriate amino acid until it reaches a stop codon and then it stops, so you have this resultant *apoB-100 protein* of 4536 aa length. In the *intestine*, however, you have a cytosine deamination (that's the editing part). If you deaminate cytosine, you get a uracil. With this you've created a stop codon in the middle, so now the protein synthetic machinery only goes up until that stop codon. This results in a protein that is less than half the size of the one made in the liver, only 2152 aa length. Remember they both come from information in the same apoB gene. So in addition to alternative splicing and alternative poly-A sites, this is another way to get more than one protein from a particular gene.

(3) What is a nonsense mutation?

With a *nonsense mutation*, the codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base—A—to become UAA, the new codon causes termination of translation at that point, and the production of a shortened (truncated) protein. The creation of a termination codon at an inappropriate place is called a "nonsense" mutation.

(1) What is a silent mutation?

With a *silent mutation*, the codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base—U—to become UCU, it still codes for serine. This is termed a "silent" mutation. This doesn't mean it's without effect, whereby there can be differences in the abundance of tRNAs coding for the same amino acid, so it can have consequence as far as rate, but the mutation will still code for the same amino acid.

(3) Describe elongation of RNA synthesis.

With the formation of each succeeding phosphodiester the enzyme moves along the DNA template strand. Complementary monomeric units are added with the elimination of pyrophosphate from the nucleoside triphosphates.

What is needed for splicing to occur?

You need specific splice sites and appropriately positioned adenosine nucleoside and you also need recognition by snRNPs, which include small noncoding RNAs in the U series.

How are miRNAs processed?

miRNAs are first transcribed as primary transcripts with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures in the cell nucleus. This processing is performed in animals by a complex, consisting of the *nuclease Drosha* and a double-stranded RNA binding protein. The pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the *endonuclease Dicer*, which also binds to an Argonaute protein and initiates the formation of the *RNA-induced silencing complex (RISC)*. This complex is responsible for the silencing of gene expression observed due to miRNAs and RNA interference.