L I F E S C I E N C E S

Transcription

1 1a L I F E S C I E N C E S 5 -UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3 5 -TTA ATA TTC GAA AGC TGC ATC GAA AAC TGT GAA TCA-3 3 -AAT TAT AAG CTT TCG ACG TAG CTT TTG ACA CTT AGT-5 NOVEMBER 2, 2006 ROBERT A. LUE

2 1a L I F E S C I E N C E S 5 -UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3 Non-template Coding Sense 5 -TTA ATA TTC GAA AGC TGC ATC GAA AAC TGT GAA TCA-3 3 -AAT TAT AAG CTT TCG ACG TAG CTT TTG ACA CTT AGT-5 Template Non-coding Anti-sense There are a variety of names used to designate the two strands of DNA that make up gene sequences. The nomenclature is related to either transcription (RNA synthesis) or to translation (protein synthesis). In the case of the former, the strand that serves as the template upon which the complementary RNA chain is polymerized is called the template strand, while the other strand is called the non-template strand. If we shift our point of reference to translation, then the non-template strand is described as the coding strand since it matches the sequence of the complementary RNA (with the substitution of uracil for thymidine), which is used to direct the assembly of the amino acid sequence in the protein. From this perspective, the template strand is therefore described as the non-coding strand. Finally, if we assume that a genetic sequence only makes sense in light of its ability to direct protein synthesis, then the coding strand could also be described as the sense strand. Consequently, from this point of view the non-coding strand could be described as the anti-sense strand

3

4 HIV Tat: Transactivating regulatory protein The Tat gene encodes an amino acid transactivator protein Enhances the rate of viral replication up to 1000-fold The protein interacts with a short sequence within the 5 LTR called TAR or Tat responsive element Only interacts with TAR in HIV RNA transcripts HIV Tat. Tat stands for transactivating regulatory protein. It s a relatively small protein, which depending on the strain of HIV, can range in size from 86 to 104 amino acids in length. In the presence of Tat, transcription of the integrated HIV genome is enhanced by up to 1000-fold. This level of transcription enhancement is essential for HIV replication. Based on what you have learned about prokaryotic and eukaryotic transcription, it would be reasonable to think of Tat as simply another transcription factor. You have seen scenarios in both prokaryotes and eukaryotes where transcription factors assemble with RNA polymerase on DNA. It is therefore not surprising to discover that Tat does bind to nucleic acids. In fact, in the 5' LTR of the viral genome there is a short sequence called TAR, with stands for Tat responsive element. However, what is surprising is that Tat does not bind to the DNA version of TAR. Tat only binds to RNA version of TAR. Furthermore, if you look at the sequence of TAR in RNA form, it forms a beautiful and quite complex hairpin structure with a bulge on one side. This is the structure that Tat binds in order to enhance transcription of the HIV genome.

5 HIV Tat enhances proviral transcription RNAP II: CTD: CycT: Cdk9: Tat: RNA polymerase II C-terminal domain Cyclin T Cyclin-dependent kinase 9 HIV Transactivator In the case of HIV Tat, we have a potent enhancer of transcription that does not bind to DNA. Instead it binds to RNA. So you might think, Isn t this a chicken and the egg problem? In other words, if Tat is supposed to enhance this transcription, which is the polymerization of RNA from a DNA template, but it only binds to the TAR sequence in the RNA form, hasn t transcription already happened? Well, in fact, the way in which it works is remarkably elegant and when first discovered revealed an entirely new way of enhancing transcription. Because Tat only binds to the RNA version of the TAR sequence it has no way to affect the initiation of transcription like the general transcription factors we discussed earlier. Instead, the initiation of HIV transcription depends on host cell transcription factors. That said, initiation is relatively inefficient, and in the absence of Tat, the production of complete RNA transcripts occurs at a low level. So, in the absence of Tat, RNA polymerase II is described as comparatively non-processive. In other words, it does not complete transcribing the entire proviral sequence and tends to fall off of the DNA. Nevertheless, a limited number of HIV RNA transcripts are made and exported to the cytoplasm for use in protein expression. One of the viral proteins expressed using these RNAs is Tat, which is then imported into the nucleus where it can enhance HIV transcription.

6 HIV Tat enhances proviral transcription Mechanism of Tat activity differs from that of typical transcription activators Tat enhances Elongation by recruiting kinases that phosphorylate RNA polymerase RNAP II: CTD: CycT: Cdk9: Tat: RNA polymerase II C-terminal domain Cyclin T Cyclin-dependent kinase 9 HIV Transactivator Although the initiation of HIV transcription is both inefficient and dependent on host cell factors, the first stretch of viral RNA synthesized happens to contain the TAR sequence. The TAR sequence folds into its elaborate hairpin structure and what you effectively get is an RNA tail that is still attached to RNA polymerase II, much the same way a mouse has a tail! Then Tat comes along and binds to the RNA tail via TAR. In addition to binding to TAR in the RNA, Tat also binds to a host cell protein named Cyclin T (CycT), which in turn binds to an enzyme named Cdk9. The enzymatic activity of Cdk9 is to promote the covalent addition of charged phosphate groups to other proteins. So, Tat effectively attaches the CycT-Cdk9 complex to the RNA tail, and brings the Cdk9 enzyme in close proximity to RNA polymerase II. This enables Cdk9 to promote the addition of phosphate groups to the domain of RNA polymerase II that we discussed earlier in the context of eukaryotic transcription. The covalent addition of phosphate groups to RNA polymerase II alters its activity and it becomes highly processive. In other words, it is now able to complete transcription and very efficiently. So Tat facilitates the nucleation or bringing together of proteins, some of which act on one another, resulting in enhanced transcription. Tat activity underscores a very important principle relevant to many biological processes: in order for a particular process or chemical reaction to occur, the various players or components must be brought together in close proximity to each other. In this case, the ability of Cdk9 to act on RNA polymerase II depends on it being close enough. And that proximity is achieved by association to Tat, which in turn is bound to the growing strand of RNA emerging from RNA polymerase II. In the absence of Tat, viral transcription is far too inefficient and new viruses will not be produced.

7 Map of the HIV genome gag pol env rev tat shared sequences split gene sequences A closer inspection of the HIV genome reveals an arrangement of genes that requires additional mechanisms for interpreting the information stored in the nucleotide sequence. Lets look at the location of five HIV genes: gag, pol, env, rev, and tat. By diagraming the location of each gene individually but maintaining the alignment at the 5 end, we can compare the stretches of nucleotide sequence that comprise each gene perhaps more easily than shown in the upper genome diagram. We immediately notice two interesting characteristics: (1) several genes appear to share stretches of nucleotide sequence (e.g. gag and pol; env, rev, and tat), and (2) some genes are split into two separate stretches of sequence. In the case of those genes that share stretches of sequence, does this mean that they have protein segments that are also identical? The answer is no, because although two HIV genes may share a stretch of nucleotide sequence, the same sequence is read differently during protein synthesis to produce two different sequences of amino acids. This is a fundamental feature of the protein synthesis process that we will discuss later in this lecture. In the case of genes that are split into two separated stretches of sequence, we will now see that the processing of the resulting RNA transcript via a mechanism known as RNA splicing allows the reassembly of a unified coding template. This is a fundamental process that also occurs for most eukaryotic genes.

8 RNA processing: the diversification of nucleic acid function 1. Eukaryotic RNAs undergo significant modification a) Eukaryotic RNAs are processed to produce functional mrna b) RNA splicing increases the diversity of eukaryotic RNAs c) Transport of RNA out of the nucleus is regulated 2. Controlling HIV protein expression by regulating RNA export Lecture Readings Alberts: pp

9 Genetic information is further diversified at the RNA and protein levels Genes are organized differently in Bacteria and Eukaryotes In prokaryotes, like bacteria, the production of so-called messenger RNA molecules which serve as the template for protein synthesis is relatively simple. The 5 end of an mrna molecule is produced by the initiation of transcription by RNA polymerase at a promoter (colored green), and the 3 end is produced by the termination of transcription. Since bacterial genes are entirely comprised of contiguous coding sequence, the bacterial protein is translated from the unprocessed RNA transcript (sometimes referred to as the primary transcript). Since bacteria lack a nucleus, transcription and translation into protein take place in a common compartment. The organization of eukaryotic genes is significantly more complex than their bacterial counterparts. The majority of eukaryotic genes are made up of sequences that encode protein and thus are expressed (so-called exons) interspersed with intervening sequences (so-called introns) that do not code for protein. Therefore, in eukaryotic cells the primary RNA transcript (sometimes referred to as the pre-mrna) 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.

10 Eukaryotic RNAs are processed to produce functional mrnas Three major RNA processing events: (1) Addition of a 5 -Cap (2) Addition of a 3 -poly(a) tail (Polyadenylation) (3) Removal of Introns (RNA Splicing) Let us consider the the three major processing steps for the primary RNA transcript of the ovalbumin gene, all of which occur in the nucleus. This gene is comprised of eight exons (colored green) and seven introns (colored yellow). As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5 end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide. The capping reaction is performed by three enzymes acting in succession: one removes one phosphate from the 5 end of the nascent RNA, another adds a GMP in a reverse linkage (5 to 5 instead of 5 to 3 ), and a third adds a methyl group to the guanosine. The cap therefore consists of an unusual 7-methyl guanosine. The 5 -cap is an identifying feature of the 5 end of eukaryotic mrnas, and helps the cell to distinguish mrnas from the other types of RNA molecules present in the cell. For example, RNA polymerases I and III produce uncapped RNAs during transcriptions. The 5 -cap also has an important role in the translation of mrnas in the cytosol as we will discuss in the next lecture. The 3 ends of eukaryotic mrnas are specified by DNA signals encoded in the genome. These DNA signals are transcribed into RNA as the RNA polymerase II moves through them, and they are then recognized by a series of RNA-processing enzymes. These enzymes cleave the RNA s sugarphosphate backbone and then add approximately 200 A nucleotides to the 3 end produced by the cleavage. This addition does not require a template; hence the poly-a tail of eukaryotic mrnas is not directly encoded in the genome. As the poly-a tail is synthesized, proteins called poly- A-binding proteins assemble onto it and, by a poorly understood mechanism, determine the final length of the tail. Poly-A-binding proteins remain bound to the poly-a tail as the mrna makes its journey from the nucleus to the cytosol and they help to direct the synthesis of a protein on the ribosome, as we will discuss in the next lecture. Finally, the intron sequences are removed and the exon sequences are re-ligated together to form a mature mrna. This process is known as RNA splicing and will be discussed later. All of the above RNA processing steps are physically coupled to transcription elongation by an ingenious mechanism. As discussed in the previous lecture, a key step of the transition of RNA polymerase II to the elongation mode of RNA synthesis is an extensive phosphorylation of the RNA polymerase II tail, called the CTD. This phosphorylation step not only dissociates the RNA polymerase II from other proteins present at the start point of transcription, it also allows a new set of proteins to associate with the RNA polymerase tail that function in pre-mrna processing. Some of these processing proteins can therefore hop from the polymerase tail onto the nascent RNA molecule to begin processing it as it emerges from the RNA polymerase. Thus, RNA polymerase II in its elongation mode can be viewed as an RNA factory that both transcribes DNA into RNA and processes the RNA it produces.

11 Nucleotide sequences determine intron boundaries R: A or G Y: C or U Three nucleotide sequences are required for splicing: 5 splice junction 3 splice junction Branchpoint adenosine Intervening intron sequence can range in length from 40 to 50,000 bases Eukaryotic cells are able to recognize and rapidly splice out intron sequences with high fidelity. The process of intron sequence removal involves three positions on the RNA: the 5 splice site, the 3 splice site, and the branch point adenosine in the intron sequence that forms the base of the excised lariat. Each of these three sites has a consensus nucleotide sequence that is similar from intron to intron, providing the cell with cues on where splicing is to take place. However, there is enough variation in each sequence to make it very difficult for scientists to pick out all of the many splicing signals in a genome sequence. Most introns range in size from about 40 nucleotides to over 50,000 nucleotides. With the exception of the three conserved sequences mentioned above, the rest of the intron sequence plays no role in the splicing process.

12 Splicing removes introns as branched lariats The branchpoint adenosine attacks the 5 splice site, cleaving the sugar phosphate backbone The 5 end of the intron is covalently linked to the adenosine, forming a loop The free 3 -OH attacks the 3 splice site, ligating the exons together and releasing the intron lariat In the first step of the splicing reaction, a specific adenosine in the intron sequence (indicated in red) attacks the 5 splice site and cleaves the sugarphosphate backbone of the RNA at this point. The cleaved 5 end of the intron becomes covalently linked to the adenine nucleotide, thereby creating a loop in the RNA molecule. This loop structure is known an the intron lariat. The released free 3 -OH end of the exon sequence then attacks the start of the next exon sequence, joining the two exons together and releasing the intron lariat. The two exon sequences thereby become joined into a continuous coding sequence and the released intron lariat is degraded.

13 Two chemical reactions remove the intron lariat 1st reaction 2 -OH of adenosine attacks the phosphate of the guanosine at the 5 splice site (donates e - ) Exchanges one bond for another 2nd reaction 3 -OH of the 5 exon attacks the phosphate of the guanosine at the 3 splice site After the intron is spliced out, the number of bonds is unchanged A single splicing event removes one intron by proceeding through two sequential phosphoryl-transfer reactions known as transesterifications. This pair of reactions join two exons while removing the intron as a lariat. Since the number of phosphate bonds remains the same, these reactions could in principle take place without a contribution of energy from ATP hydrolysis. However, the machinery that catalyzes pre-mrna splicing is complex and hydrolyzes many ATP molecules per splicing event. This complexity is presumably needed to ensure that splicing is highly accurate, while also being sufficiently flexible to deal with the enormous variety of introns found in a typical eukaryotic cell. Frequent mistakes in RNA splicing would severely harm the cell, as they would result in malfunctioning proteins.

14 RNA splicing is executed by the Spliceosome complex The Spliceosome is a large assembly of discrete small nuclear ribonucleoprotein particles (snrnps), each made up of proteins and small nuclear RNAs Structural rearrangements within the Spliceosome depend on base-pairing between snrnas and the pre-mrna The transcription and subsequent processing of mrna primarily depends on the activity of many proteins. In contrast, RNA splicing largely depends on RNA molecules instead of proteins. RNAs recognize intron-exon borders and catalyze the chemistry of splicing. These RNA molecules are relatively short (less than 200 nucleotides each), and are known as snrnas (small nuclear RNAs). Each snrna is complexed with at least seven protein subunits to form a snrnp (small nuclear ribonucleoprotein). snrnps form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mrna splicing in the cell. The spliceosome is a remarkably dynamic macromolecular machine. It is assembled on pre-mrna from separate components, and parts enter and leave it in an orderly fashion as the splicing reaction proceeds. During the splicing reaction, recognition of the 5 splice junction, the branch point adenosine site and the 3 splice junction is performed largely through base-pairing between various snrnas and the consensus RNA sequences in the pre-mrna. In the course of splicing, the spliceosome undergoes several shifts in which one set of base-pair interactions is broken and another is formed in its place. For example, U1 is replaced by U6 at the 5 splice junction. This type of RNA-RNA rearrangement, in which the formation of one RNA-RNA interaction requires the disruption of another, occurs several times during the splicing reaction. It permits the checking and rechecking of RNA sequences before the chemical reaction is allowed to proceed, thereby increasing the accuracy of splicing. As noted earlier, although ATP hydrolysis is not required for RNA splicing per se, it is required for the stepwise assembly and rearrangements of the spliceosome. Some of the additional proteins that make up the spliceosome use the energy of ATP hydrolysis to break existing RNA-RNA interactions so as to allow the formation of new ones. In all, more than 50 proteins, including those that form the snrnps, are required for each splicing event. Once the splicing reaction is completed, the snrnps remain bound to the lariat and the spliced intron is released. The disassembly of these snrnps from the lariat requires another series of RNA-RNA rearrangements dependent on ATP hydrolysis, thereby allowing the snrnas to recycle for another round of splicing.

15 RNA splicing is executed by the Spliceosome complex Many spliceosome components reside in discrete nuclear bodies called speckles The dynamic behavior of speckles is linked to transcription (activity of RNA Pol II) David Spector Some of the many components that make up the spliceosome are thought to reside in discrete domains in the nucleus called speckles. As the name implies, nuclei that are stained with fluorescently-labeled antibodies that bind to certain snrnps display a speckled pattern. These nuclear speckles may function as storage depots fro spliceosome components and display remarkably dynamic behaviors that appear to be directly coupled to sites of transcription. As the above time lapse videos demonstrate, individual speckles appear to extend and retract material in a dynamic fashion, with the extension events spatially coupled with sites of RNA polymerase II activity. Thus it would appear that activated RNA polymerase II somehow triggers the recruitment of snrnp components from these nuclear speckles. The time lapse videos were created by David Spector using living cells transfected with an snrnp protein fused to GFP.

16 RNA processing: the diversification of nucleic acid function 1. Eukaryotic RNAs undergo significant modification 2. Controlling HIV protein expression by regulating RNA export a) HIV Rev accelerates the nuclear export of selected viral RNAs b) Switching from early expression of regulatory proteins to the late expression of structural proteins and enzymes Lecture Readings Alberts: pp

17 HIV Rev: regulator of viral protein expression Essential for the control of HIV RNA splicing HIV RNAs exit the nucleus - Unspliced Single-spliced Double-spliced Rev enhances the amount of unspliced and single-spliced HIV RNA transcripts available in the cytoplasm for translation Like Tat, the HIV Rev protein is essential for viral replication, but unlike Tat it affects which viral proteins are made in the cytoplasm. Rev stands for regulator of viral protein expression. However, Rev does not affect protein translation directly, rather it affects the splicing of HIV RNA. How does Rev control RNA splicing? Does it block splicing in some cases? Is there some way in which it either activates splicing or inhibits splicing? I mentioned the possibility of alternative splicing earlier in this lecture, and HIV mrnas are very interesting examples of alternative splicing. I will refer to these HIV RNAs as mrnas (or messenger RNAs) because they are all translated in the cytoplasm and therefore function as the information-rich message that dictates what proteins are made. That said, three differently spliced HIV mrnas can leave the nucleus and be available for protein translation in the cytoplasm. You can have HIV mrnas that aren t spliced at all -- they are described as unspliced. You can have HIV mrnas that have been spliced once (single-spliced), so one intron has been removed. Finally you can have HIV mrnas that have been spliced twice (double-spliced), so two introns have been removed. And as one might imagine, the proteins that are expressed from each of those different HIV mrnas are different. So this is in part how you obtain the range of proteins that are produced by the virus from a 9.8 kb genome. The ability to generate alternative RNA splicing patterns is very important for both HIV and many eukaryotic genes. For well over a decade, it was not clear how Rev controlled which particular spliced form of HIV mrna was made available in the cytoplasm. Was it an actual direct interaction with the splicing machinery of the nucleus or was it something else? In fact, what we now know is that Rev exerts its effect via transport of HIV mrna. Rev promotes the transport of the unspliced and single-spliced HIV RNA transcripts from the nucleus (highlighted in the diagram). This enables Rev to control which viral proteins are subsequently synthesized or translated in the cytoplasm.

18 Nuclear export of HIV RNA Rev protein RRE Rev response element Early gene products Double-spliced RNAs produce viral regulatory proteins including Rev Late gene products Single & Unspliced RNAs produce structural and enzymatic components of HIV Rev acts at a very important stage of the HIV replication cycle. It determines what viral mrnas actually exit the nucleus and therefore what mrnas are available, either for translation or for packaging into new viruses. In the absence of Rev, you do not get expression of many key viral proteins that are required for new viruses especially the structural proteins. So how does Rev work? Rev protein binds to a particular RNA sequence called RRE, the Rev response element. RRE is found in the sequence of the HIV genome, but as one might expect, Rev only binds to the RNA form of RRE following transcription. In the early stages of the HIV replication cycle when Rev is absent, you observe weak HIV transcription that produces a small number of fulllength RNA transcripts. All of these transcripts are double-spliced (two introns are removed) to produce a short 2 kb viral mrna. This 2 kb mrna is transported from the nucleus by inefficient means and is available for translation in the cytoplasm. The protein products of this small mrna include the Rev and Tat proteins. As time passes, both Rev and Tat are transported into the nucleus and affect RNA splicing and transcription respectively. In the case of Rev it binds to those HIV RNA transcripts that still have the RRE sequence. Since RRE is contained in one of the introns, the only RNAs that still have the RRE sequence are those that are either unspliced or single-spliced. Double-spliced RNAs therefore lack RRE and are not affected by Rev. Rev specifically binds to the unspliced and the single-spliced transcripts, which in the presence of Rev, are the mrna transcripts found predominantly in the cytoplasm. Unlike the double-spliced mrna, which produced the regulatory Tat and Rev proteins, the unspliced and single-spliced mrnas produce the structural and enzymatic components of HIV, such as matrix proteins and the HIV protease enzyme. Rev is an example of an early gene product, and needs to accumulate to a sufficient concentration to promote the export of unspliced and the single-spliced mrna. Once this occurs, you observe a switch from early to late gene products with the latter translated from unspliced and single-spliced transcripts in the cytoplasm. Rev therefore controls the switch from early to late expression of HIV genes.

19 HIV Rev mechanism of action XPO = Exportin Nuclear transport receptor that facilitates export through nuclear pores Ran = Protein that regulates XPO activity Rev coopts the XPO+Ran complex So how does the fact that Rev binds to unspliced and single-spliced mrnas get them out of the nucleus? Once again it is a case of bringing together various proteins required for a particular process. In the case of Rev, the viral protein binds to the RRE sequence and also binds to a host cell protein named Exportin. Exportin is an example of a nuclear transport receptor that facilitates the export of material out of the nucleus. It actually enables cargo molecules, including RNAs and proteins, to pass through nuclear pores and into the cytoplasm. Exportin, in turn, is controlled by a regulatory protein by the name of Ran. Simply put, Rev serves to connect the unspliced and the single-spliced HIV mrnas to the Exportin-Ran complex inside the nucleus. Rev therefore links HIV RNAs to the normal nuclear export pathway. This linkage literally accelerates the export of viral RNAs from the nucleus, specifically those that still contain RRE, namely the unspliced and the single-spliced forms. One can think of Rev activity as a race against splicing. In other words, Rev accelerates the export of HIV RNA such that there is not enough time for complete splicing (double-splicing) to occur in the nucleus. This is why only double-spliced HIV mrnas reach the cytoplasm when Rev is absent the HIV transcripts remain in the nucleus long enough to have both introns spliced out. Once the HIV mrna plus Rev-Exportin-Ran complex reaches the cytoplasm, Ran undergoes a conformational change that breaks down the complex. This releases the HIV mrna into the cytoplasm for translation, and Rev, Exportin, and Ran are separately imported (recycled) back into the nucleus via a different class of proteins called Importins. You should remember that Exportin and Ran are entirely normal host cell proteins that also export host cell RNAs. Studying how HIV Rev works, actually allowed researchers to discover the activity of Exportin and to figure out how host cell RNAs are transported across the nuclear envelope. So this was a clear case in which understanding how a component of a pathogen works, revealed a critical pathway that works for normal processes in the cell.

20

21 Break Out A1 Protein A1 and Protein C bind to RNA in the nucleus Experiment: Human cells are fused with frog cells, producing hybrid cells containing nuclei from both species Protein synthesis is blocked with a drug After two hours the cells are simultaneously labeled with different fluorescent antibodies that bind to human A1 (red) and human C (green) C (A) (B) (C) The two nuclei in the hybrid cell have also fused The antibodies that bind to human A1 also bind to frog A1 Human A1 is subject to both nuclear export and import unfused human cell unfused frog cell hybrid cell Break Out followup: Protein A1 and C are known components of heterogenous ribonucleoprotein particles or hnrnps. These complexes are formed in the nucleus and consist of multiple hnrnp proteins (such as A1 and C) complexed with pre-mrna. These proteins are thought to play an important role in the packaging and export of mrna from the nucleus. Lets consider the three interpretations presented. Interpretation A does not seem likely because one would expect to see both protein A1 and C present in both nuclei of the hybrid cell. In other words, if the nuclei of the hybrid cell had been imaged in the process of fusion, which could account for the A1 staining in both as their contents mix, then the staining for C would likewise be visible in both based on the simple mixing of nuclear contents. Interpretation B does not seem likely because the unfused frog cell in the lower left does not exhibit any staining with the antihuman A1 antibody. This unfused cell serves as a negative control to confirm that both antibodies do not recognize frog proteins. Therefore, interpretation C appears to be the most likely since the only way both nuclei can enhibit staining for A1 is if the protein were exported from the human nucleus and imported into the frog nucleus. This also confirms the recycling of protein A1 in that the very same molecules of A1 that are exported are subsequently competent for import. The use of cycloheximide to block protein synthesis was important for this conclusion since no new molecules of A1 could have been synthesized in the cytoplasm and subsequently imported.

22 Translation: the RNA-directed synthesis of proteins 1. The role of RNA in protein synthesis a) Three classes of RNA are required to synthesize proteins b) mrnas are decoded in sets of three nucleotides c) The structure and function of transfer RNA d) Proofreading by aminoacyl-trna synthetase 2. The translation machinery and cycle Lecture Readings Alberts: pp McMurry: pp

23 Three classes of RNA are required to synthesize proteins Messenger RNA (mrna) serves as the informational template Transfer RNA (trna) are molecular adaptors that match amino acids to the mrna code Ribosomal RNA (rrna) associate with proteins to form the ribosome The ribosome is a macromolecular machine consisting of proteins and RNA Ribosome model with trna and rrna Decodes the mrna and promotes the polymerization of amino acids into proteins Protein translation is the whole process by which the nucleotide sequence of an mrna is used to order and to join the amino acids in a protein. DNA stores the information-rich code required for protein synthesis and RNA is the molecular intermediary that carries out the instructions encoded in DNA. On the other hand, most of the activities in the cell are carried out by proteins. As Dan pointed out earlier, proteins are the macromolecular workhorses of the cell and the accurate synthesis of proteins is essential for the proper functioning of cells and organisms. Dan showed you that the linear order of amino acids in each protein determines its three-dimensional structure and activity. This means that the assembly of amino acids in their correct sequence, as encoded in DNA, is necessary for the proper synthesis of functional proteins. Three kinds of RNA molecules perform different but cooperative functions in protein synthesis: 1. Messenger RNA (mrna) carries the genetic information copied from DNA in the form of a series of three-base or triplet code each of which specifies a particular amino acid. 2. Transfer RNA (trna) is the molecular adaptor that deciphers the triplet code in mrna. Each type of amino acid has its own type of trna, which binds it and escorts it to the growing end of a polypeptide chain if called for by the next triplet on the mrna. The correct trna with its attached amino acid is selected based on it having a three-base sequence that can base-pair with its complementary triplet in the mrna. 3. Ribosomal RNA (rrna) associates with a set of proteins to form ribosomes. These large macromolecular complexes catalyze the polymerization of amino acids into protein chains. They also bind trnas and various accessory molecules necessary for protein translation. As we will discuss shortly, the three types of RNA participate in translation in all cells with each of their distinct functions playing an essential role in the process.

24 mrna sequences are decoded in sets of three nucleotides The Genetic Code Each nucleotide triplet in mrna is called a codon Codons are read consecutively 5 to 3 on the mrna Four nucleotides gives 4 3 or 64 possible codon triplets Most amino acids are encoded by several codons 3 codons encode a stop signal As David discussed earlier in the course, RNA is made up of ribonucleotides (adenine, cytidine, guanine, and uracil), while DNA contains deoxyribonucleotides of adenine, cytidine, guanine, and thymine. Since four nucleotides, taken individually, could represent only 4 of the 20 possible amino acids in coding the linear arrangement in proteins, a group of nucleotides is required to represent each amino acid. The code employed must be capable of specifying at least 20 amino acids. If two nucleotides were used to code for one amino acid, then only 16 (or 4 2 ) different code units could be formed, and there would not be sufficient unique codes to account for 20 amino acids. However, if a group of three nucleotides is used for each amino acid, then 64 (or 4 3 ) code units are available for use. Therefore, any code using groups of three or more nucleotides will have more than enough units to encode 20 amino acids, and many such arrangements are mathematically possible. The actual genetic code used by all cells is a triplet code, with every three nucleotides being decoded from a specified starting point in the mrna and exclusively in the 5 to 3 direction. Each triplet is called a codon. Of the 64 possible codons in the genetic code, 61 specify individual amino acids, which indicates that many amino acids are encoded by more than one codon. The remaining three available codon sequences are used to signal the end or termination of the translation process (stop signals). Only two methionine and tryptophan have a single codon; at the other extreme, leucine, serine, and arginine are each specified by six different codons. The different codons for a given amino acid are said to be synonymous. The code itself can be termed degenerate since it contains redundancies.

25 mrna sequences can be decoded in three different reading frames mrna code can be translated in one of three reading frames Each reading frame is defined by the starting position of the first codon Each protein is translated in a specific reading frame When considering how a sequence of triplet codons can be read to determine the sequence of a linear chain of amino acids, it is important to remember that this genetic code does not have inserted punctuation. In other words, once the first codon position has been defined, all of the other codons are defined in a contiguous and continuous sequence with no breaks or interruptions. The phasing of the codons defined by the first is known as the reading frame of that particular mrna transcript. As shown above, this also means that a particular mrna theoretically could be translated in three different reading frames because each reading frame is determined by the starting nucleotide of the first codon. The different reading frames define different sequences of codons which will yield different amino acid sequences. The vast majority of mrnas are read in only one frame because stop codons encountered in the other two possible reading frames terminate translation before a functional protein is produced. This accounts for the impact of many genetic mutations that either insert or remove (delete) a single base-pair in DNA -- the resulting shift in the phasing of codons on the 3 side of the change alters the effective reading frame and an inappropriate sequence of amino acids is encoded. On the other hand, some mrnas contain overlapping information that can be translated in different reading frames, yielding different protein sequences. This amounts to overloading a single nucleotide sequence with the code for more than one protein and is one way in which HIV manages to produce so many viral proteins from a relatively small (9.8 kb) genome. The mechanism for altering the reading frame for a same sequence is beyond the scope of this course but suffice it to say, it often requires variations in which nucleotide is defined as the beginning of the first codon.