Protein Synthesis in Prokaryotes and Eukaroytes

Transcription and synthesis of different RNAs
Processing of RNA transcript
Catalytic RNA
RNA splicing and Spliceosome
Transport of RNA through nuclear pore
Translation and polypeptide synthesis
Posttranslational modification
Protein trafficking and degradation
Antibiotics and inhibition of protein synthesis.

1. Protein Synthesis in P k s & E k s The simple Story that started complex LIFE!! Prepared by BIR BAHADUR THAPA CDB, TU NEPAL Guided by Dr. GIRI PD. JOSHI CDB, TU NEPAL

2. About picture, which did not tell itself is: - A non-coding RNA ( ncRNA ) is an RNA molecule that is not translated into a protein . The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene . Abundant and functionally important types of non-coding RNAs include transfer RNAs ( tRNAs ) and ribosomal RNAs ( rRNAs ), as well as small RNAs such as microRNAs , siRNAs , piRNAs , snoRNAs , snRNAs , exRNAs , scaRNAs and the long ncRNAs such as Xist and HOTAIR . Non-coding RNAs contribute to diseases including cancers , autism , and Alzheimer's . Many of the newly identified ncRNAs have not been validated for their function . It is also likely that many ncRNAs are non functional (sometimes referred to as Junk RNA ), and are the product of false transcription .

3. About sub-heading “ The simple Story that started LIFE !!” The origins of life? The central dogma is so central to all living things, but one wonders how it may have evolved. Life requires both storage and replication of genetic information, and the ability to catalyze specific reactions. RNA has both of these abilities. RNA thought to be the original molecule of life, carrying both genetic info and performing chemical reactions (ribozymes ). Life then shifted to a DNA platform for the storage of the genetic information because of its increased chemical stability and double-stranded format that enables proofreading Life then shifted to a protein platform for chemical processes ->broader chemical functionality

4. Contents under Protein synthesis in Prokaryotes and Eukaryotes : Transcription and synthesis of different RNAs P rocessing of RNA transcript Catalytic RNA RNA splicing and Spliceosome T ransport of RNA through nuclear pore Translation and polypeptide synthesis Posttranslational modification P rotein trafficking and degradation A ntibiotics and inhibition of protein synthesis.

5. Transcription and synthesis of diff. RNAs 1. Transcription : DNA to mRNA

6. Transcription and synthesis of diff. RNAs What is transcription? RNA synthesis (=Transcription) is the process of copying information in DNA sequences into RNA sequences . How is transcription different from replication of DNA? DNA replication serves to copy all the genetic material of the cell and occurs before a cell divides. Transcription copies short stretches of the coding regions of DNA to make RNA. Different genes may be copied into RNA at different times in the cell's life cycle . What enzyme carries out transcription? This process is catalyzed by the enzyme RNA Polymerase . "RNA polymerase" is a general term for an enzyme that makes RNA. There are many different RNA polymerases. The basic transcription process is more or less similar in prokaryotes and eukaryotes, though the regulation of transcription is much more elaborate in eukaryotes .

7. Transcription and synthesis of diff. RNAs How does RNA Polymerase carry out its function? To carry out RNA synthesis, all RNA Polymerases, prokaryotic and eukaryotic. must do the following: 1. Search the DNA template for promoters (sites on the DNA where the polymerase binds to start transcription.) 2. Interact with other proteins that regulate transcription. 3. Unwind a short stretch of the DNA to expose single stranded DNA to copy into RNA 4. Select the correct RNA nucleotides, based on the DNA sequence, and assemble the RNA chain. 5 Recognize termination signals and stop synthesizing RNA when a termination signal is detected. How is RNA Polymerase like DNA Polymerase? Like DNA polymerase, RNA Polymerase synthesizes new strands only in the 5' to 3' direction. How is RNA Polymerase different from DNA Polymerase? RNA Polymerase doesn't require a primer to start making RNA. RNA Polymerase uses ribonucleotides , not deoxyribonucleotides .

8. Different RNAs The major RNAs can be assigned to three major classes: ( 1) The cytoplasmic messenger RNAs ( mRNAs ) and their nuclear precursors ( pre-mRNAs ) carry the information that is used to specify the sequence, and therefore ultimately the structure, of all proteins in the cell. ( 2) Other RNAs do not encode protein but function directly, playing major roles in various metabolic pathways, including protein synthesis. - These include the ribosomal RNAs ( rRNAs ) and transfer RNAs ( tRNAs ), which are the key components of the protein synthesis machinery; - the small nuclear RNAs ( snRNAs ), which form the core of the pre-mRNA splicing system; & - the small nucleolar RNAs ( snoRNAs ), which are important factors in ribosome biogenesis. These RNAs are generally much longer-lived than mRNAs and therefore often are referred to as stable or noncoding RNAs ( ncRNAs ).

9. Transcription and synthesis of diff. RNAs (3) The third and most recently identified class of RNA comprises several structurally related groups of very small (21 to 25 nucleotides) RNA species that play important roles in regulating gene expression. Base pairing between endogenous micro-RNAs ( miRNAs ) and target mRNAs in the cytoplasm represses their translation into protein. The packaging of DNA into a nontranscribed form termed heterochromatin is promoted by a class of nuclear, small centromeric RNAs ( ncRNAs ). The introduction of small double-stranded RNAs into many cell types and organisms results in cleavage of the target mRNA and consequent silencing of gene expression. This phenomenon is described as RNA interference ( RNAi ), and the RNAs are referred to as small interfering RNAs ( siRNAs ). In addition, a heterogeneous set of longer ncRNAs ( lncRNAs ) have been implicated in a variety of nuclear events. How does an RNA polymerase know where to start copying DNA to make a transcript? Signals in DNA indicate to RNA polymerase where it should start and end transcription. These signals are special sequences in DNA that are recognized by the RNA polymerase or by proteins that help RNA polymerase determine where it should bind the DNA to start transcription. A DNA sequence at which the RNA polymerase binds to start transcription is called a promoter .

10. Transcription and synthesis of diff. RNAs What does a promoter look like in prokaryotes? A typical prokaryotic promoter has three recognizable elements: The transcription start site (this the base in the DNA across from which the first RNA nucleotide is paired). Sequences that are before the start site are said be "upstream" sequences. The -10 sequence : this is a 6 bp region centered about 10 bp upstream of the start site. The consensus sequence at this position is TATAAT (i.e., this is the sequence found at this position in the majority of promoters studied) 3. The -35 sequence : this is a 6 bp sequence at about 35 basepairs upstream from the start of transcription. The consensus sequence at this position is TTGACA. The sequences at -10 and -35 are recognized and bound by the RNA polymerase before transcription can begin.

11. Transcription and synthesis of diff. RNAs How does RNA polymerase bind and carry out transcription in prokaryotes ? Prokaryotic RNA polymerases have 2 components, a core enzyme and a sigma factor ( a.ka . sigma subunit) . The sigma factor is necessary for the RNA polymerase to bind tightly to the promoter and initiate transcription. Once transcription starts, the sigma factor falls off, and the core enzyme continues copying the DNA into RNA till it reaches a terminator . A terminator is a sequence of DNA that signals RNA polymerase to stop transcribing.

12. Transcription and synthesis of diff. RNAs In what ways does transcription differ in prokaryotes and eukaryotes? 1 . In eukaryotes, the DNA template exists as chromatin, not as free DNA. The packaging of the DNA must therefore be "opened up" to allow access for transcription. We have already considered how chromatin remodeling complexes and histone modifications can make DNA regions accessible for transcription. 2 . Eukaryotes have three RNA polymerases, not one as in bacterial cells. 3. All three eukaryotic RNA polymerases need additional proteins to help them get transcription started. In prokaryotes, RNA polymerase by itself can initiate transcription. 4. In addition to promoters, eukaryotic genes often have extra regulatory sequences many kilobases away from the transcription start site. In bacteria, regulatory sequences are generally adjacent to the gene that they control. 5. In eukaryotic cells, transcription is separated in space and time from translation: - Transcription happens in the nucleus , and the RNAs produced are processed further before they are sent into the cytoplasm. - Protein synthesis (translation) happens in the cytoplasm . In prokaryotic cells, RNAs can be translated as they are coming off the DNA template, and because there is no nucleus, transcription and protein synthesis occur in a single cellular compartment .

13. Eukaryotic Protein Synthesis vs. Prokaryotic Protein Synthesis Eukaryotic Protein Synthesis mRNA molecules are  monocistronic , containing the coding sequence only for one  polypeptide .  protein synthesis occurs in the  cytoplasm .  most of the gene have  introns or non-coding sequences along with exons  or  coding sequences. The exons are joined together and introns are removed during mRNA processing. The primary mRNA transcript  undergoes  processing  and splicing to change into a functional mRNA. mRNA molecules are modified by the addition of a 5’G cap formed of methylated  guanosine  triphosphate.  A poly A tail formed of about 200 adenine nucleotides is added at the 3’end of  mRNA . 5’cap initiates translation by binding the  mRNA to small ribosomal subunit usually at the first codon AUG. The first amino acid methionine entering the ribosome is  not  formylated . The pre imitation complex formation is initiated by nine initiated factors.  No. of initiating factors is much more than prokaryotes. About ten IFs have been identified in reticulocytes an RBC. These are  eIF1, eIF2, eIF3, eIF4 , eIF5, eIF6 ,eIF4B, eIF4C,eIF4D, eIF4F small subunit of ribosome (40 S) gets dissociated with the initiator amino acyl  tRNA  ( MettRNA  Met) without the help of mRNA. The complex joins mRNA later on. Prokaryotic Protein Synthesis mRNA molecules are polycistronic  containing the coding sequence of several genes of a particular metabolic pathway.  protein synthesis begins even before the transcription of mRNA molecule is completed. This is called coupled transcription - translation.  do not have introns (Except Archaebacteria ). Therefore  mRNA processing  is not required. splicing of mRNA  transcript does not occur.  No such cap is formed at 5’end of bacterial mRNA. No poly A  tail is added to bacterial mRNA.  translation begins at an AUG codon preceded by a special nucleotide sequence.  The first amino acid methionine is  formylated  into N  formyl  methionine. Only two initiating factors are involved. Three initiating factors found in prokaryotes.  PIF-1 , PIF-2 , PIF-3  30 S subunit first complexes with mRNA (30S-mRNA) when then joins with f Met  tRNA  f-

14. Transcription and synthesis of diff. RNAs What do the three RNA Polymerases do? RNA polymerase I- transcribes ribosomal RNA genes RNA polymerase II- transcribes protein coding genes (that is, it makes mRNA) RNA polymerase III- transcribes transfer RNA genes. We will focus on RNA Polymerase II (sometimes referred to as Pol II) which transcribes messenger RNAs. As always, Pol II must find and bind a promoter to initiate transcription. What does a eukaryotic promoter look like? Eukaryotic promoters have some recognizable features: 1. The start site for transcription. 2. The TATA-box : This is a sequence about 25 basepairs upstream of the start of transcription. 3. Variable numbers of upstream elements: these are short DNA sequences that are within 100 bp upstream of the start of transcription.

15. Transcription and synthesis of diff. RNAs What are the additional proteins needed to start transcription? General transcription factors are proteins that help eukaryotic RNA polymerases find transcription start sites and initiate RNA synthesis. For RNA polymerase II these transcription factors are named TFIIA, TFIIB and so on (TF= transcription factor, II=RNA polymerase II, and the letters distinguish individual transcription factors). The complex composed of RNA polymerase and the general transcription factors bound at the TATA box is called the basal transcription complex or transcription initiation complex .  It is the minimum requirement for any gene to be transcribed.

16. Processing of RNA transcript The newly made RNA, also known as the primary transcript (the product of transcription is known as a transcript ) is further processed before it is functional. Both prokaryotes and eukaryotes process their r & t-RNAs . The major difference in RNA processing, however, between prokaryotes and eukaryotes, is in the processing of mRNAs . in bacterial cells, the mRNA is translated directly as it comes off the DNA template. In eukaryotic cells, RNA synthesis, which occurs in the nucleus, is separated from the protein synthesis machinery, which is in the cytoplasm. In addition, eukaryotic genes have introns, noncoding regions that interrupt the gene. The mRNA copied from genes containing introns will also therefore have noncoding regions that interrupt the information in the gene. These noncoding regions must be removed before the mRNA is sent out of the nucleus to be used for protein synthesis. The process of removing the introns and rejoining the coding sections or exons, of the mRNA, is called splicing . Once the mRNA has been capped, spliced and had a polyA tail added, it is sent from the nucleus into the cytoplasm for translation. The lifespan of RNAs varies greatly. Some RNAs are longlived , others are rapidly degraded. RNA degradation is another step at which gene expression can be regulated after the initial transcriptional control step.

17. Processing of RNA transcript What are the processing steps for messenger RNAs? Messenger RNAs are processed in eukaryotic cells, not in bacterial cells. The three main processing steps are 1 . Capping at the 5' end 2 . Addition of a polyA tail at the 3' end and 3 . Splicing to remove introns

18. Processing of RNA transcript What is the initial transcript of mRNA called ? The initial product of transcription of an mRNA is called the pre-mRNA or primary transcript. After it has been processed and is ready to be exported from the nucleus, it is called the mature mRNA or processed mRNA . What happens in the capping step? In the capping step of mRNA processing, a 7-methyl guanosine is added at the 5' end of the mRNA.

19. Processing of RNA transcript What is the function of capping? The cap protects the 5' end of the mRNA from degradation by nucleases and also helps to position the mRNA correctly on the ribosomes during protein synthesis .   What happens at the 3' end of the mRNA? The 3' end of a eukaryotic mRNA is first trimmed, then an enzyme called PolyA Polymerase adds a "tail" of about 200 As to the 3' end.   What is the function of the tail? Evidence indicates that the polyA tail plays a role in efficient translation of the mRNA, as well as in the stability of the mRNA. The cap and the polyA tail on an mRNA are also indications that the mRNA is complete (i.e., not defective). How are introns removed from the pre-mRNA? Introns are removed from the pre-mRNA by the activity of a complex called the spliceosome . The spliceosome is made up of proteins and small RNAs that are associated to form protein-RNA enzymes called small nuclear ribonucleoproteins or snRNPs (pronounced SNURPS ).

20. Processing of RNA transcript What signals indicate where an intron starts and ends? The base sequence at the start (5' end) of an intron is GU while the sequence at the 3' end is AG . There is also a third important sequence within the intron, called a branch point, that is important for splicing.

21. Processing of RNA transcript The newly made RNA, also known as the primary transcript (the product of transcription is known as a transcript ) is further processed before it is functional. Both prokaryotes and eukaryotes process their r & t-RNAs . The major difference in RNA processing, however, between prokaryotes and eukaryotes, is in the processing of mRNAs . in bacterial cells, the mRNA is translated directly as it comes off the DNA template. In eukaryotic cells, RNA synthesis, which occurs in the nucleus, is separated from the protein synthesis machinery, which is in the cytoplasm. In addition, eukaryotic genes have introns, noncoding regions that interrupt the gene. The mRNA copied from genes containing introns will also therefore have noncoding regions that interrupt the information in the gene. These noncoding regions must be removed before the mRNA is sent out of the nucleus to be used for protein synthesis. The process of removing the introns and rejoining the coding sections or exons, of the mRNA, is called splicing . Once the mRNA has been capped, spliced and had a polyA tail added, it is sent from the nucleus into the cytoplasm for translation. The lifespan of RNAs varies greatly. Some RNAs are longlived , others are rapidly degraded. RNA degradation is another step at which gene expression can be regulated after the initial transcriptional control step.

22. Catalytic RNA cRNA are RNA molecules that have enzyme activity. The classic example is the hammerhead ribozyme. Catalytic RNAs are involved in a number of biological processes, including RNA processing and protein synthesis. Discovery of catalytic RNA contributed to the hypothesis of an 'RNA world', describing the origin of life as starting from RNA. Some RNA molecules can function like enzymes and exert a catalytic action on themselves or on other molecules, known as Catalytic RNA, presenting a new target for drugs and can be used for inactivating unwanted RNA or DNA molecules by a specific cleavage reaction .

23. Catalytic RNA Discovery- 1980s Until recently, protein enzymes were thought to be the only biologically active catalysts. It was believed that DNA stored the genetic information and that RNA played the role of an intermediate courier between the genetic messages contained in the DNA and the ribosomes, where proteins are synthesized. Other RNAs, like transfer RNAs ( tRNAs ) and ribosomal RNAs ( rRNAs ), were considered as helper molecules to assist the function of proteins. Today, RNA molecules are the only molecules known both to store genetic information (as in the RNA viruses and viroids or during transport in the form of mRNA ) and to exert biological catalysis . nondispensable biological activity - In the tRNA -processing enzyme ribonuclease P ( RNase P), the RNA moiety contains the catalytic activity . Self-splicing introns often occur within ribosomal genes, as in the rRNA

24. Catalytic RNA Very recently, it has been established that protein synthesis on the ribosome is catalysed by the 23S ribosomal RNA compound and , thus , that the ribosome is actually a ribozyme ( Nissen et al ., 2000). Ribozymes need to acquire three-dimensional architectures to promote specific interactions with cofactors , especially divalent metal ions, and other functional domains for processing RNA substrates. Ribozymes are generally built up of several structural subdomains made of helical segments connected by tertiary contacts. - Functional regions are usually located in single-stranded regions , such as internal loops or bulges. - Specific tertiary contacts occur between hairpin and internal loops, especially those positioned on the outside of the molecule . - The subdomains have various functions and are responsible for substrate recognition, specific sequence alignment and catalytic activity, leading to a modular and hierarchically organized architecture .

25. Catalytic RNA Some ribozymes, like the hammerhead ribozyme, the hairpin ribozyme and the RNAase P RNA, are under extensive clinical research for their ability to cleave other specifically chosen substrate RNAs. The therapeutic applications range from cleavage of viral RNAs, like the acquired immune deficiency syndrome (AIDS)- causing human immunodeficiency virus (HIV) RNA, to silencing of carcinogenic or mutated cellular RNAs, or the control of gene expression in vivo. Secondary structure of a minimal hairpin ribozyme with substrate RNA bound. Circles represent individual nucleotides and lines indicate canonical (Watson-Crick) base pairs Predicted secondary structure and sequence conservation of the HH9 ribozyme found conserved from lizard to human genomes.

26. Catalytic RNA Sequence and Structure RNA molecules are built up of RNA helices interlinked by internal loops and multiple junctions. Double-stranded helical regions are formed through standard Watson–Crick base pairs. One groove of the standard type A-form double-stranded RNA helix is deep and narrow, while the other one is shallow and wide. Double-stranded regions mainly act as a framework or rigid spacer to organize and to orientate other structural and functional recognition elements . Helices can be connected by three-, four-or multi-way junctions, which form a sequence-dependent joint , which is either flexible or fixed, depending, among other factors, on the type and concentration of metal ions . Single-stranded regions comprise a whole range of structural elements, including base bulges, internal loops or bubbles and terminal or hairpin loops. These can simply serve as flexible linkers between helical domains, or they interact with other bases, usually forming non-Watson–Crick pairs to widen or tighten the grooves or to induce kinks and bends in the RNA backbone.

27. Catalytic RNA Catalytic Mechanisms RNAase P RNA- an endoribonuclease responsible for the maturation of the 5′ termini of the majority of all known tRNAs in all cell types studied to date, involves in a site-specific hydrolysis. The enzyme is conserved in all three kingdoms—bacteria, archaea , and eukarya —with mitochondria and chloroplasts having activities separate from the nucleus in eukaryotes . Eukaryotes also contain a second RNA-protein enzyme, called RNase MRP, which is closely related to RNase P. The RNA components share common structural features, and the complexes share eight common proteins. RNase MRP cleaves the pre- rRNA between the small and large subunit rRNAs . The biological reactions catalysed by ribozymes mainly involve phosphodiester cleavage or transfer (i.e. relegation to another nucleotide). The 2’ ribose hydroxyl group, characteristic of RNA, is always directly or indirectly involved in catalysis. Ribozymes can be considered as metalloenzymes . Indeed , the folding and/or catalytic activities of ribozymes often depend on the presence of and interaction with divalent metal ions (Pyle, 1993).

28. Catalytic RNA Classes of Catalytic RNAs tRNA processing by ribonuclease P RNA- a true recycling catalyst is the ubiquitous endonuclease enzyme processing precursor tRNA 5’ ends. Functional groups involved in catalysis are the 2’ ribose hydroxyl group of the conserved pyrimidine (usually a U) at the position preceding the cleavage and a highly conserved purine at the position of the cleavage on the pre- tRNA . Intron Splicing- All three major types of cellular RNAs, tRNA , rRNA and mRNA , are known to contain intervening sequences (IVS ) or introns, which have to be precisely excised to produce functional molecules or the right reading frame for protein synthesis . This process, known as RNA splicing, requires the recognition of the 5’ and 3’ exonic sequences, strand cleavage and ligation. Although most eukaryotic introns are spliced by a complex machinery, the spliceosome , some introns in bacteriophages or organelles self-splice. Small Catalytic RNAs- simplest, e ach undergoing self cleavage and has a well-defined tertiary structure in the presence of divalent cations . The hammerhead ribozyme- smallest known ribozyme . All conserved bases are in the single-stranded regions linking the helical stems and terminal loops add stability and ensure correct folding. The three dimensional structure obtained by X-ray diffraction studies ( Pley et al., 1994) shows that most of the nucleotides in the single-stranded regions base-pair to form a Y-shaped structure. The hairpin ribozyme- second smallest catalytic RNA molecule with a functional length of 50 nucleotides . It catalyses not only a cleavage reaction but also its reverse reaction (ligation). 4. Artificial Ribozymes- Natural catalytic RNAs are limited to reactions involving phosphoryl centres . artificial ribozymes have been selected by in vitro selection which can yield ribozymes using different catalytic metal ion cofactors . Ribozymes with ribosyl -cleavage properties , a ligase activity (3’–5’,2’–5’ and 5’–5’ phosphodiester linkage ) and with a phosphorylation reaction could be selected.

29. Catalytic RNA Ribozymes: Catalytic RNAs that cut things, make things, and do odd and useful jobs- Walter aand Engelke , 2002 Catalytic RNAs, or ribozymes, are a fossil record of the ancient molecular evolution of life on Earth and still provide the essential core of macromolecule synthesis in all life forms today. Are they also an avenue to the development of new catalysts to recreate evolution, or to use as therapeutics and molecule sensors ? (Walter & Engelke , 2002) The original discovery of ribozymes by Cech and Altman (Nobel laureate- 1990) was twofold – RNA segments that cut themselves out of larger RNAs (self-splicing introns) and a protein-assisted RNA enzyme ( ribonuclease P) that cuts the leader sequences off all transfer RNAs throughout the three organismal domains . Universal use of RNA in synthesis of macromolecules The use of RNA in protein synthesis has long been part of the central dogma. Not only is information carried in messenger RNA (mRNA) triplet codons, but transfer RNA ( tRNA ) serves as the adapter to interpret codons into amino acids; and the enormously complex ribosome , containing both ribosomal RNA ( rRNA ) and protein components, serves as the factory for decoding the message into protein chains .

30. Catalytic RNA Ribozyme Gene Therapy- Lewin & Hauswirth , 2001 RNA enzymes – ribozymes – are being developed as treatments for a variety of diseases ranging from inborn metabolic disorders to viral infections and acquired diseases such as cancer. Ribozymes can be used both to down regulate and to repair pathogenic genes. In some instances, short-term exogenous delivery of stabilized RNA is desirable, but many treatments will require viral-mediated delivery to provide long-term expression of the therapeutic catalyst. Current gene therapy applications employ variations on naturally occurring ribozymes, but in vitro selection has provided new RNA and DNA catalysts, and research on trans-splicing and RNase P has suggested ways to harness the endogenous ribozymes of the cell for therapeutic purposes . There are two basic modes for therapy that targets the genetic basis of disease: replace or resect. For diseases caused by recessive mutations, gene therapists try to complement the defective gene. For dominant disease mutations, however, introducing a normal gene will not work. Many of these diseases are associated with hyperactivation (in the case of oncogenes ), or aggregation of a mutant protein (in the case of neurodegenerative diseases ), with a normal copy of the same gene being present on the partner chromosome . In these cases, expression of the defective gene must be silenced or at least limited. For such autosomal dominant genetic diseases, ribozymes are a particularly appealing tool: they can be used to reduce expression of a pathogenic gene by digesting the mRNA it encodes .

31. Catalytic RNA Ribozyme Gene Therapy- [ Lewin & Hauswirth , 2001]

32. Catalytic RNA Ribosome is a Ribozyme- Cech , 2000 The AA we obtained by digestion of steak, salmon or lettuce salad are loaded onto tRNAs and rebuilt into proteins in the ribosome, the cell’s macromolecular protein synthesis factory…. Its Key component are so highly conserved among all of the Earth’s species that a similar entity must have fueled protein synthesis in the LUCA i.e., L ast U niversal C ommon A ncestor i.e. ancestor of all extant life. Ribosome task- simple one i.e. joining multiple AA through peptide linkage but it also performs the remarkable task of choosing the AA to be added to the growing polypeptide chain by reading successive mRNA.

33. Splicing: pre-mRNA to mRNA RNA splicing and Spliceosome

34. RNA splicing and Spliceosome Spliceosomes are dynamic macromolecular multimegadalton organelles, a endogenous RiboNucleoProteinComplexes (RNPC) composed of small nuclear RNA ( snRNA ) & approximately 100 other associated proteins with a ribozyme at its core, that remove intervening noncoding regions- introns in protein-encoding genes of a precursor messenger RNA ( pre-mRNA). The assembly depends on both complementary base pairing between the small nuclear RNAs and the intron and exon substrates, and on extensive protein–RNA and protein–protein interactions. These interactions are fundamental for proper spliceosomal function. At the core of the spliceosome are five small nuclear RNAs ( snRNAs ) termed U1, U2, U4, U5, and U6. U1-U5 snRNAs are produced by RNA polymerase II transcription and are 5′- capped with 7-methylguanosine, while U6 is transcribed by RNA Pol III and has a different cap structure . Each of the snRNAs is associated with a set of 8 Sm proteins, B/B′, D3, D2, D1, E, F, and G . The Sm proteins bind to each other and to a highly conserved sequence on U1, U2, U4, and U5 snRNAs . The association of Sm proteins and snRNAs occurs in the cytoplasm in a controlled and precise order, and the 5′cap structure of snRNAs must be hypermethylated before the small nuclear ribonucleoprotein ( snRNP ) complex is imported back into the nucleus. U6 snRNA diverges from this assembly pathway and associates to Sm -like proteins LSm2, LSm3, LSm4, LSm5, LSm6, LSm7, and LSm8 in the nucleus.

35. RNA splicing and Spliceosome How does splicing actually take place? There are two main steps in splicing- 1 st step , the pre-mRNA is cut at the 5' splice site (the junction of the 5' exon and the intron). The 5' end of the intron then is joined to the branch point within the intron. This generates the lariat-shaped molecule characteristic of the splicing process. 2 nd step , the 3' splice site is cut, and the two exons are joined together, and the intron is released . https://www.youtube.com/watch?v=FVuAwBGw_pQ

36. RNA splicing and Spliceosome

37. RNA splicing and Spliceosome If there are multiple exons in an mRNA can they be spliced in different combinations? Yes, different combinations of exons can be spliced together to generate different mature mRNAs (recall that this is called alternative splicing ).

38. RNA splicing and Spliceosome What is the advantage of alternative splicing? Alternative splicing allows the production of many different proteins using relatively few genes, since a single RNA with many exons can, by mixing and matching its exons during splicing, create many different protein coding messages . Because of alternative splicing, each gene in our DNA gives rise, on average, to three different proteins . What happens to RNAs after they have fulfilled their functions? All RNAs are eventually degraded in the cell. Ribosomal RNAs and transfer RNAs, which are in constant use in the cell for protein synthesis, are relatively long-lived. The stability of messenger RNAs varies widely. Some are degraded rapidly, while others may have a longer half-life. The level of a particular mRNA is determined by both the rate of its synthesis and the rate of its breakdown by the cell. This provides the cell with an additional point of control for gene expression.

39. Nuclear Pore Complexes Nuclear Pore Complexes : Small-sized nuclear pores keep most large molecules from entering the nucleus. But some large protein molecules are required in the nucleus and enter it though a larger complex protein structure called the nuclear pore complex (NPC). This complex consists of at least 456 protein molecules. It surrounds each pore and allows larger molecules to exit or enter by expanding the pore openings to a larger size. NPC's cross the double-walled nuclear membrane, allowing the transport of water-soluble molecules through the membrane. Nuclear pore- Side view. 1. Nuclear envelope. 2 . Outer ring. 3 . Spokes. 4 . Basket. 5 . Filaments.

40. Transport of RNA through nuclear pore Since the DNA is transcribed into mRNA in the nucleus, and protein synthesis takes place in the cytoplasm, the mRNA has to exit the nucleus to the cytoplasm. The environment in the nucleus differs in many ways from that of the cytoplasm. To separate these two environments from each other the nucleus is enclosed by a double membrane, and the only connection to the surrounding cytoplasm is through channels called the nuclear pore complex (NPC ). When it is time for the mRNA to leave the nucleus, the mRNA is believed to be "tagged" by proteins which serve as export signals, directing the mRNA to the nuclear pore complex that the mRNA is to leave. The mRNA and it's bound export proteins then attaches to export receptors and the whole complex (RNA, export signal proteins and export receptors) is translocated through the nuclear pore complex. The mRNA is released into the cytoplasm and is immediately ready for the next step: translation »

41. Transport of RNA through nuclear pore a.  The mRNA molecule is transported to the nuclear pore. Before the translocation through the nuclear pore begins, some proteins, for example the splicing components, disassociate from the mRNA. b.   Export proteins bind to the mRNA and as a first step in the translocation, the mRNA docks with the cage-like structure of the nuclear pore complex . c .  The mRNA is translocated through the nuclear pore with the 5' end, the CAP structure, in the lead. The translocation through the nuclear pore is an energy requiring process, but the mechanism for the transport is not known. d .  When the mRNA arrives at the cytoplasmic side of the nuclear pore, even if parts of the RNA are still within the pore or on the nuclear side, many proteins disassociate from the mRNA. Among those are the export proteins which return to the nucleus. The mRNA is immediately ready for the next step: translation

42. Transport of RNA through nuclear pore a.  The mRNA molecule is transported to the nuclear pore. Before the translocation through the nuclear pore begins, some proteins, for example the splicing components, disassociate from the mRNA. b.   Export proteins bind to the mRNA and as a first step in the translocation, the mRNA docks with the cage-like structure of the nuclear pore complex. c.  The mRNA is translocated through the nuclear pore with the 5' end, the CAP structure, in the lead. The translocation through the nuclear pore is an energy requiring process, but the mechanism for the transport is not known . d.  When the mRNA arrives at the cytoplasmic side of the nuclear pore, even if parts of the RNA are still within the pore or on the nuclear side, many proteins disassociate from the mRNA. Among those are the export proteins which return to the nucleus. The mRNA is immediately ready for the next step: translation

43. Translation and Polypeptide synthesis Translation: mRNA to tRNA

44. Translation and Polypeptide synthesis Once mRNA has passed out of the nuclear pore, it determines the synthesis of a polypeptide . A ribosome attaches to the starting codon, at one end of an RNA molecule. The tRNA molecule with a complementary anticodon sequence moves to the ribosome,  and pairs up with the sequence on the mRNA. A tRNA molecule with a complimentary anticodon pairs up with the next codon on the mRNA. The ribosome moves along the mRNA, bringing together two tRNA molecules, each pairing up with the corresponding two codons on the mRNA. By means of an enzyme, ATP and and two amino acids on the tRNA are joined by a peptide bond. The ribosome moves onto the third codon, on the mRNA linking the amino acids. As this happens the first tRNA is released from its amino acid, from the amino acid pool in the cell. This process continues until a complete polypeptide chain is created.

45. Translation and Polypeptide synthesis 1. Initiation Translation initiation operates through a 30S initiation complex (30S-IC), consisting of the 30S, mRNA, initiator fMet-tRNA , and three initiation factors, IF1, IF2, and IF3. Subsequently, the 30S-IC associates with the 50S, which r eleases the Initiation Factors ( IFs ) and leaves the initiator- tRNA at the peptidyl - tRNA -binding Site (P site), base-paired to t he start Codon of The mRNA .

46. Translation and Polypeptide synthesis 2. Elongation The elongation phase involves the movement of tRNAs in a cyclic fashion through the three tRNA -binding sites (A→P→E) on the ribosome. The first step in the cycle involves the delivery of the aminoacyl-tRNA ( aa-tRNA ) to the aa - tRNA -binding site (A site), which is facilitated by the elongation factor EF-Tu•GTP . Hydrolysis of GTP by EF- Tu leads to its dissociation from the ribosome, allowing aa-tRNA accommodation. Peptide-bond formation then proceeds, transferring the entire polypeptide chain from the P- tRNA to the aa-tRNA in the A site. The ribosome now has a peptidyl-tRNA at the A site and an uncharged tRNA at the P site. This ribosomal state is highly dynamic with the tRNAs oscillating between classical (A and P sites) and hybrid states (A/P and P/E sites on 30S/50S). EF-G binds to the ribosome, which stabilizes the tRNAs in hybrid states, hydrolyzes GTP to GDP, and catalyzes the translocation reaction. Translocation shifts the peptidyl-tRNA from the A/P hybrid state to the P site and the deacylated tRNA from the P/E to the exit site (E site). EF-G•GDP dissociates leaving the A site vacant for the next incoming aa-tRNA .

47. Translation and Polypeptide synthesis 3. Termination/Recycling Arrival of an mRNA stop codon in the A site of the ribosome signals the termination of protein synthesis. Release factor 1 (RF1) or RF2 binds to the ribosome and hydrolyzes the peptidyl-tRNA bond, releasing the translated polypeptide chain from the ribosome. RF1/2 is recycled from the ribosome by RF3 in a GTP-dependent fashion. The ribosome is then split into subunits by the concerted action of EF-G and ribosome recycling factor (RRF), thus recycling the components for the next round of translation.

48. Translation and Polypeptide synthesis tRNAs attach to the correct amino acid in the cytoplasm – directed by enzymes 2. Small subunit of the ribosome binds to the 5’ cap of the mRNA

49. Translation and Polypeptide synthesis 3. The first tRNA attaches to the first three nucleotides of the mRNA The first three nucleotides of every mRNA is AUG . This codes for the amino acid Methoinine .

50. Translation and Polypeptide synthesis 4. The large subunit slides into place so the tRNA is in the middle slot of the ribosome, the P site .

51. Translation and Polypeptide synthesis 5. The next tRNA with the appropriate amino acid enters the A site matching to the mRNA codon . - Determine the amino acid coming in based on the mRNA codon sequence – use mRNA codon chart.

52. Translation and Polypeptide synthesis 6. The amino acid on the tRNA in the P site attaches to the amino acid on the tRNA in the A site and forms a peptide bond .

53. Translation and Polypeptide synthesis 7. The ribosome shifts down the mRNA causing the tRNAs (which are still attached to the mRNA) to translocate (move) into the adjacent site on the ribosome . - the tRNA in the A site moves to the P site - the tRNA in P site moves to the E site and then leaves

54. Translation and Polypeptide synthesis 8. This process (steps 5 – 7) repeats over and over building the amino acid chain until a STOP codon is reached . - STOP codons = UGA, UAG, UAA - bring in a protein called a Release Factor and this causes the amino acid chain (protein) to be freed and the ribosome to detach from the mRNA.

55. Translation and Polypeptide synthesis What is peptide: A Simple Introduction of Peptide Synthesis. Peptides and proteins are linear polymers of amino acids linked by amide "peptide bonds" (Fig. 1). The peptide bond is formed by linking an amino group to a caroboxyl group on another amino acid. Amino acids are primary amines that contain an alpha carbon that is connected to an amino (NH3) group, a carboxyl group (COOH), and a variable side group (R). Carboxylic acid and carboxylate groups are normally not very reactive. It requires activating the carboxylic acid for the formation of the amide peptide bond.

56. Translation and Polypeptide synthesis

57. Translation and Polypeptide synthesis

58. Post-translational modification (PTM) Newly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications before they reach their final destinations: 1. Formation of disulfide bonds 2. Proper folding 3. Addition and processing of carbohydrates 4. Specific proteolytic cleavages 5. Assembly into multimeric proteins The most common are:- -specific cleavage of precursor proteins; -formation of disulfide bonds; or - covalent addition or removal of low molecular weight groups thus leading to modifications like Phosphorylation, Acetylation, N-linked glycosylation, Amidation , Hydroxylation, Methylation, O-linked glycosylation, Ubiquitylation , Pyrrolidone Carboxylic Acid, Sulfation

59. Post-translational modification (PTM) Disulfide Bonds Are Formed and Rearranged in the ER Lumen Correct Folding of Newly Made Proteins Is Facilitated by Several ER Proteins Assembly of Subunits into Multimeric Proteins Occurs in the ER Only Properly Folded Proteins Are Transported from the Rough ER to the Golgi Complex Many Unassembled or Misfolded Proteins in the ER Are Transported to the Cytosol and Degraded ER-Resident Proteins Often Are Retrieved from the Cis -Golgi

60. Post-translational modification (PTM) PTM is a biochemical modification that occurs to one or more amino acids on a protein after the protein has been translated by a ribosome, the covalent and generally enzymatic modification of proteins following protein biosynthesis . Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling . Fundamental role- Regulating the folding of proteins, their targeting to specific subcellular compartments, their interactions with ligands or other proteins, and their functional states, such as catalytic activity in the case of enzymes or the signaling function of proteins involved in signal transduction pathways. Study Method- PROTEOMICS i.e. systemic identification of proteins from cells and tissues that involves separation of proteins and their isoforms by size or charge heterogeneity by 2- dimensional gel electrophoresis, recovery of the individual spots from gel by mass spectrometry. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one.

61. Post-translational modification (PTM) Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation , which can promote protein folding and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation , often targets a protein or part of a protein attached to the cell membrane . Varying degrees of posttranslational modification affect the hydrophobicity by adding a glycophospholipid to the C-terminal carboxyl group of the protein(s). The lipid allows the enzyme to be tethered to plasma membranes. Other forms of post-translational modification consist of cleaving peptide bonds , as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification . For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds .

62. Post-translational modification (PTM) Glycosylation is a posttranslational modification whereby one or more sugar groups are covalently linked to a target protein forming a glycoprotein. These sugars are often essential for the proper structure and function of the protein. Some types of post-translational modification are consequences of oxidative stress . Carbonylation is one example that targets the modified protein for degradation and can result in the formation of protein aggregates. Specific amino acid modifications can be used as biomarkers indicating oxidative damage .

63. Protein Trafficking and Degradation

64. Antibiotics and Inhibition of Protein Synthesis. A protein synthesis inhibitor is a substance that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins . any antibiotic , usually refers to any substances that act at the ribosome level (either the ribosome itself or the translation factor ), taking advantages of the major differences between prokaryotic and eukaryotic ribosome structures .

65. Antibiotics and Inhibition of Protein Synthesis. Inhibitors of both Prokaryotic and Eukaryotic Protein Synthesis: Aurintricarboocylic acid inhibits formation of the initi­ation complex by preventing the association of mRNA with the small ribosomal subunit. Edeine , a polypeptide isolated from Bacil­lus brevis , inhibits the binding of aminoacyl-tRNA and N- formylmet - tRNA M f et (in prokaryotes) to the small subunit . Fusidic acid is a steroidal antibiotic; in pro­karyotes, it inhibits the binding of aminoacyl-tRNA to the ribosome, whereas in eukaryotes, it inhibits trans­location by reacting with elongation factor . Puromycin , mimics aminoacyl-tRNA and binds to the free A site of ribosomes engaged in protein synthesis. Catalytic formation of a bond between the nascent polypeptide and puromycin is followed by the release of the peptidyl-puromycin from the ribosome, as no further elongation is possible.

66. Antibiotics and Inhibition of Protein Synthesis. 2. Inhibitors Specific for Prokaryotes : Chloramphenicol ( Chloromycetin ) binds to the large subunit of prokaryotic ribosomes and interferes with the functioning of peptide synthetase , thereby inhibit­ing chain elongation. Colicin E3 inhibits protein syn­thesis in prokaryotes by interfering in some manner with the functioning of the small subunit. Erythromy­cin binds to ribosomes that are not engaged in protein synthesis, preventing their potential participation, but does not bind to ribosomes containing nascent chains (i.e., ribosomes that are part of a functioning polysome ). Streptomycin binds to protein S12 of the small ribosome subunit, causing release of N- formylmet - tRNA M f et from initia­tion complexes (thereby preventing initiation of chain growth) and also causing misreading of the codons of mRNA by ribosomes already involved in chain elonga­tion.

67. Antibiotics and Inhibition of Protein Synthesis. 3. Inhibitors Specific for Eukaryotes : Anisomycin is an antibiotic produced by Streptomyces that inhibits peptide bond formation when bound to the small ribosomal subunit. Cycloheximide binds to the large subunit, preventing the translocation of tRNA in the A site to the P site. Diphtheria toxin (produced by a strain of Corynebacterium diphtherial ) inhibits protein synthesis through its action on EF-2 ( translocase ). Ricin acts on the large subunit, preventing formation of the 80 S initiation complex. Sparsomycin , produced by Streptomyces , inhibits the association of the amino acid moi­ety of aminoacyl-tRNA from binding to the large subunit and, in so doing, blocks peptide synthetase . THchodermin is the only chemical compound so far identified as a specific inhibitor of the termination stage of polypeptide synthesis.

68. Antibiotics and Inhibition of Protein Synthesis. 4. Inhibitors of Organeiiar Protein Synthesis : C hloramphenicol , a strong inhibitor of prokaryote protein synthesis, blocks synthesis in mito­chondria and chloroplasts, whereas cycloheximide , which blocks eukaryote cytoplasmic ribosomal protein synthesis, is without effect on mitochondrial and chlo­roplast synthesis . These observations provided added credence for the notion that prokaryotic cells, mito­chondria, and chloroplasts have a common evolution­ary origin. It is now clear, however, that the picture is considerably more complex. For example, streptomy­cin, which inhibits prokaryotic protein synthesis, fails to inhibit mitochondrial protein synthesis in yeast cells . Erythro­mycin inhibits the synthesis of proteins in prokary­otes, yeast mitochondria, and chloroplasts but fails to block protein synthesis in mammaliam mitochondria.

69. Antibiotics and Inhibition of Protein Synthesis. 4. Inhibitors of Organeiiar Protein Synthesis: The nature of mitochondrial protein synthesis varies among differ­ent groups of eukaryotes. M itochondria from higher eukaryotes are more resistant to inhibi­tors of prokaryotic protein synthesis than are mito­chondria from lower eukaryotes . In chloroplasts, pro­tein synthesis is inhibited by the same agents that block this process in prokaryotic cells .

70. Antibiotics and Inhibition of Protein Synthesis. 4. Inhibitors of Organeiiar Protein Synthesis: The differential sensitivity of eukaryotic cytoplas­mic and mitochondrial ribosomes to specific inhibitors provides a means for examining the sources of certain mitochondrial proteins. The synthesis of a mitochon­drial protein in the presence of cycloheximide (a cyto­plasmic inhibitor) indicates that the mitochondria are the source of the protein, whereas synthesis of the protein in the presence of chloramphenicol indicates that the mitochondrial protein is produced in the cyto­plasm and then moves to the mitochondria .

71. Antibiotics and Inhibition of Protein Synthesis. 1. PCN/pen derived from Penicilium notatum Penicillin   is a group of antibiotics which include  - penicillin G (intravenous use),  - penicillin V (oral use ), - procaine penicillin etc .. degrades the cell wall of bacteria which is made up of peptidoglycan without interfering with the host cell wall

72. Antibiotics and Inhibition of Protein Synthesis. 2. Streptomycin Derived from actino -bacterium Streptomyces griseus ( G+ve bacteria; with high G and C content in DNA ) Trisaccharide Medically important from aminoglycosides family At higher concentration binds with fMet-tRNA to ribosomes and inhibit initiation At lower concentration it leads to the misreading of genetic code

73. Antibiotics and Inhibition of Protein Synthesis. 2. Tetracycline Broad spectrum four ring compound produced from Streptomyces Blocks A site on the ribosomes so that binding of aminoacyl-tRNA is inhibited. 3. Chloramphenicol Inhibits the peptidyl transferase activity on 70s ribosomes Act at low concentration Doesn’t affect cytosolic protein synthesis

74. Antibiotics and Inhibition of Protein Synthesis. 4. Cycloheximide Inhibitor of protein synthesis in eukaryotes Toxic to human and used in in-vitro research application Exerts its effect by interfering with translocation step thus blocking translational elongation used as fungicide 5 . Erythromycin Binds to 50s subunit of prokaryotes and blocks translocation step thereby freezing the peptidyl – tRNA in the A site

75. Antibiotics and Inhibition of Protein Synthesis. Stages- Protein Synthesis Antibiotics Mechanism Earlier Rifamycin stage- inhibits prokaryotic DNA transcription into mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit. Initiation Linezolid probably by preventing the formation of the initiation complex . Ribosome assembly Neomycin prevents ribosome assembly by binding to the prokaryotic 30S ribosomal subunit . Aminoacyl tRNA entry Tetracyclines , Tigecycline block the A site on the ribosome, preventing the binding of aminoacyl tRNAs . Proofreading Aminoglycosides , interfere with the proofreading process, causing increased rate of error in synthesis with premature termination. Peptidyl transfer Chloramphenicol blocks the peptidyl transfer step of elongation on the 50S ribosomal subunit in both bacteria and mitochondria . Macrolides bind to the 50s ribosomal subunits, inhibiting peptidyl transfer . Ribosomal translocation Macrolides , clindamycin , , aminoglycoside ribosomal translocation inhibition Termination Macrolides and clindamycin cause premature dissociation of the peptidyl-tRNA from the ribosome

76. References Walter, F., & Westhof , E. (2001). Catalytic RNA. eLS . Walter, N. G., & Engelke , D. R. (2002). Ribozymes: catalytic RNAs that cut things, make things, and do odd and useful jobs. Biologist (London, England) , 49 (5), 199. Pley HW, Flaherty KM and McKay DB (1994) Three-dimensional structure of a hammerhead ribozyme. Nature 372: 68–74. Pyle AM (1993) Ribozymes: a distinct class of metalloenzymes . Science 261: 709–714. ltman S. Nobel lecture. Enzymatic cleavage of RNA by RNA. Biosci Rep. 1990; 10:317–337 . [ PubMed: 1701103] Butcher SE. Structure and function of the small ribozymes. Curr Opin Struct Biol. 2001; 11:315–320 . [ PubMed: 11406380] Cech TR. Nobel lecture. Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena. Biosci Rep. 1990; 10:239–261. [PubMed: 1699616] Cech TR. The ribosome is a ribozyme. Science. 2000; 289:878–879. [PubMed: 10960319 ]

77. References http:// oregonstate.edu/instruction/bi314/fall11/geneexpression.html https:// www.sciencedirect.com/topics/neuroscience/spliceosome https:// themedicalbiochemistrypage.org/rna.php#editing www.biologydiscussion.com/rna/rna-introduction-synthesis-and-types/11775 https:// www.youtube.com/watch?v=FVuAwBGw_pQ https://www.nobelprize.org/educational/medicine/dna/a/transport / https:// sciencing.com/mrna-leave-nucleus-10050146.html https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC3770912/pdf/nihms413112.pdf https:// en.wikipedia.org/wiki/Post-translational_modification https://www.ncbi.nlm.nih.gov/books/NBK21741 / http:// chemistry.elmhurst.edu/vchembook/584proteinsyn.html