Mol Bio L4

• Gene expression is the translation of information encoded in a gene into protein or RNA structures.
• Gene expression involves two steps transcription and translation.
• Expressed genes include genes transcribed into mRNA and then translated into protein, and genes transcribed into RNA (rRNAs, tRNA, ncRNA`s) without translation into protein.

Transcription

• The process of making a copy of genetic information stored in a DNA strand into a complementary strand of messenger RNA (mRNA) with the aid of RNA polymerases.
• Transcription includes initiation, elongation and termination.
• During the initiation of transcription, the cluster of proteins assembles on the promoter sequence at the upstream (5') end of a gene to form the transcription initiation complex.
• RNA polymerase II forms a transcription bubble and begins polymerization of ribonucleotides (rNTPs) at the start site, which is located within the promoter 5`UTR region.
• In eukaryotes, RNA polymerase II initiates transcription in the vast majority of protein coding genes.
• RNA polymerase I initiates transcription in rRNA precursor coding gene.
• RNA polymerase III initiates transcription in other RNA coding genes.
• In prokaryotes exist one RNA polymerase with less complex structure.
• Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription bubble.
• Transcription initiation is complete when the first two ribonucleotides (rNTP) are linked by a phosphodiester bond.
• During the stage of strand elongation, RNA polymerase moves along the template DNA one base at a time, opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind it.
• One ribonucleotide at a time is added to the 3` end of the growing RNA chain during strand elongation by the protein complex polymerase.
• Approximately eight nucleotides at the 3` end of the growing RNA strand remain base-paired to the template DNA strand in the transcription bubble.
• The synthesis of the RNA transcript proceeds in a 5 - 3 direction.
• The strand of the gene which is transcribed and serves as template is read in 3 - 5 direction.
• Transcription continues through both exonic and intronic regions of the gene in the presence of elongation factors that stimulate elongation.
• The elongation complex, comprised of RNA polymerase, template DNA, and the nascent RNA strand, is very stable.
• RNA synthesis occurs at a rate of approximately 1000 nt/min at 37°C.
• The elongation complex must remain intact for more than 24 hours.
• During transcription termination the completed RNA molecule, or primary transcript, is released from the RNA polymerase.
• The polymerase dissociates from the template DNA.
• The AAUAAA sequence is a signal to terminate transcription.

RNA Processing

• The primary transcripts of protein-coding genes are precursor mRNAs (pre-mRNAs) that must undergo several modifications, termed RNA processing, to form a functional mRNA.
• At the 5 end of a growing RNA chain, appearing from the surface of RNA polymerase II, is immediately acted on by several enzymes that together synthesize the 5 cap (7-methylguanylate) called capping.
• The cap protects mRNA from enzymatic degradation, assists in export to cytoplasm, and has a role in initiation of translation in cytoplasm.
• Processing at the 3` end of a pre-mRNA involves cleavage by an endonuclease to provide a free 3'-OH group.
• Adenosines are then added by an enzyme called poly(A) polymerase.
• RNA splicing is the final step in the processing of mRNA molecules.
• Splicing involves the internal cleavage of the transcript to remove the introns and ligation of the coding exons.
• Splicing is ensured by protein complex – spliceosome.
• Alternative splicing is a process in which exons within a pre-mRNA transcript are differentially joined or skipped.
• This results in multiple protein isoforms being encoded by a single gene.
• Alternative splicing generates proteomic diversity in humans.
• It also significantly affects various functions in cellular processes such as tissue specificity, developmental states, and disease conditions.
• This mechanism increases the informational diversity and functional capacity of a gene during post-transcriptional processing.
• All post-transcriptional processes, as well as splicing, take place in the nucleus.
• The matured mRNA is formed as the result, which is subsequently exported through the nuclear pore complex to the cytoplasm.
• Highly selective transport of mRNA from the nucleus to the cytoplasm is closely coupled to correct RNA processing.
• Gene expression differs in prokaryotes and eukaryotes.
• Gene expression in prokaryotes and eukaryotes is spatially distinct and ensured by slightly different mechanism.

Translation

• A step in protein biosynthesis, wherein the coded information carried by mRNA is decoded to produce the specific sequence of amino acids in a polypeptide chain.
• Translation involves initiation, elongation and termination.

rRNA and Ribosomes

• The ribosome is an RNA-protein complex, found in the cell, that directs elongation of a polypeptide at a rate of 3 - 5 amino acids added per second.
• Ribosomes physically move along an mRNA molecule and catalyse the assembly of amino acids into polypeptide chains.
• Ribosomes also bind tRNAs and various accessory proteins necessary for protein synthesis.
• In all cells, each ribosome consists of a large and a small subunit.
• The two subunits contain rRNAs of different lengths, as well as a different set of proteins.
• Transfer RNA (tRNA) is the key to decoding the codons in mRNA.
• Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain.
• Each specific tRNA molecule contains a three-nucleotide sequence, called an anticodon, that can base-pair with its complementary codon in the mRNA.
• The tRNA molecule is chemically linked to a particular amino acid via a high-energy bond to participate in protein synthesis.
• To participate in protein synthesis, a tRNA molecule must become chemically linked to a particular amino acid via a high-energy bond, forming an aminoacyl-tRNA.
• The anticodon in the tRNA base-pairs with a codon in mRNA.
• The activated amino acid can then be added to the growing polypeptide chain.
• The process is ensured by enzyme – aminoacyl-tRNA-synthetase.
• Translation begins with the binding of the small ribosomal subunit to a specific sequence on the mRNA chain.
• The small subunit binds via complementary base pairing between one of its internal subunits and the ribosome binding site which consists of a ~10 nt sequence on the mRNA, approx. 5 to 11 nt upstream the initiating codon, AUG.
• A specific tRNA molecule (tRNA-Met) recognizes and binds to the initiator codon (AUG) – P site.
• All other tRNAs bind to A site of ribosome.
• The large subunit binds, forming the initiation complex.
• The process is mediated by initiation factors (proteins).
• The initiation Met is subsequently removed from the polypeptide sequence.
• Correct positioning of the initiation complex is followed by stepwise addition of amino acids by the in-frame translation of the mRNA.
• This process requires elongation factors, entry of each succeeding aminoacyl-tRNA, formation of a peptide bond, and the movement of the ribosome one codon at a time along the mRNA.
• The STOP codon is a signal for termination of translation.
• The process is assisted by two release factors.
• Translation completes the flow of genetic information within the cell.
• The sequence of nucleotides in DNA has now been converted to the sequence of amino acids in a polypeptide chain.
• Translation of ribosomes differs between prokaryotes and eukaryotes.

Proteins

• Protein function is derived from its three-dimensional structure.
• Three-dimensional structure is specified by the amino acid sequence.
• Protein primary structure is the amino acids linked by peptide bonds into a linear chain, based on mRNA sequence.
• Protein secondary structure exhibits various spatial arrangements resulting from the folding of localized parts of a polypeptide chain, and it is stabilized by hydrogen bounds.
• Stabilized protein secondary structures include alpha-helix, beta-sheet structures, random coils and turns,
• Protein tertiary structure, the overall conformation of a polypeptide chain, is the three-dimensional arrangement of all its amino acid residues.
• Structure is primarily stabilized by hydrophobic interactions between the nonpolar side chains.
• Structure is primarily stabilized by hydrogen bonds between polar side chains, and by peptide bonds.
• These stabilizing forces hold together elements of secondary structure.
• Proper folding of proteins within cells is mediated by proteins called chaperones.
• Chaperones bind to the amino (N) terminus of the growing polypeptide chain.
• This stabilizes the unfolded configuration until synthesis of the polypeptide is completed.
• The completed protein is then released from the ribosome and is able to fold into its correct three-dimensional conformation.
• Some enzymes involved in protein folding includes isomerases, which catalyse protein folding by breaking and re-forming covalent bonds,
• Isomerases also form disulphide bonds between cysteine residues.
• Isomerases catalyse the isomerization of peptide bonds between proline residues.
• Isomerases are important in stabilizing the folded structures of many proteins.
• An important step in the maturation of many proteins is cleavage of the polypeptide chain called proteolysis.
• Proteolytic modifications play role in the translocation of many proteins across membranes.
• This achieved by cleavage of amino-terminal sequences.
• Cleavage of larger precursors is important to activate enzymes or hormones, such as activation of insulin.
• In eukaryotic cells proteins are modified by the addition of sugar a process called glycosylation.
• Glycosylation is initiated in the endoplasmic reticulum before translation is complete.
• Some proteins in eukaryotic cells are modified by the attachment of lipids to the polypeptide chain.
• Improperly folded or modified proteins are often degraded in degradation pathways.

Human Protein-Folding Diseases

• Errors in protein folding may lead to human pathology.
• al-antitrypsin deficiency (MIM #613490) where the secretion of al-antitrypsin, a secreted protease inhibitor, is changed and/or misfolded.
• The changed and misfolded al-antitrypsin subsequently leads to lung damage and liver damage.
• Misfolded proteins are not degraded, and accumulates in the ER of hepatocytes, which is the site of synthesis leading to liver damage.
• al-antitrypsin is meant to inhibit the action of proteases in the lung, so failure to maintain the protease inhibitor results in extensive damage to the lung's connective tissue.
• Amyloid accumulation refers to abnormal fibrous, extracellular, protein deposits found in organs and tissues.
• Amyloid is insoluble and is structurally dominated by ẞ-sheet structure.
• Amyloid fibrils are formed by normally soluble proteins, which assemble to form insoluble fibers that are resistant to degradation.
• The formation of amyloid fibrils can accompany disease, and each disease is characterized by a specific protein or peptide that aggregates.
• Huntington's disease (MIM #143100) is a neurodegenerative disorder that is caused by an unstable expansion of a CAG repeats within the coding region of the HTT gene.
• This results in an elongated stretch of glutamine near the N- terminus of the protein.
• Huntingtin fragment length and amount, along with the glutamine repeat, are critical factors in determining the aggregation process.