From Gene to Protein

1. From Gene to Protein Protein Structure and Folding

2. What is a gene? • A unit of heredity that is transferred from a parent to offspring and is held to determine some characteristic • Proteins coded directly by genes • A distinct sequence of nucleotides forming part of a chromosome “The basic physical unit of heredity; a linear sequence of nucleotides alongasegment of DNA thatprovides the coded instructions for synthesisofRNA,which, whentranslated into protein, leads to the expression of hereditary character”.

5. The 21st and 22nd genetically encoded amino acids • Selenium is an essential nutrient for many organisms including humans. • The major biological form of selenium is a component of the amino acid selenocysteine. • Encoded in > 15 genes in prokaryotes that are involved in redox reactions, and in > 40 genes in eukaryotes that code for various antioxidants and the type I iodothyroninedeiodinase of the thyroid gland. • Selenoproteinsare essential for mammalian development. • The UAG codon in some instances can trigger incorporation of pyrrolysine rather than termination. • The 22nd amino acid, pyrrolysine, was recently identified in a few archaebacteria and eubacteria. In the archaebacteriumMethanocarcinabarkeri, pyrrolysine has been found in some methylamine methyltransferases. required to generate methane from methylamines

6. Modified Nucleotides and Codon Bias • The degeneracy of the code results in some amino acids being coded for by as many as six codons (e.g. leucine, serine), whereas others are coded for by as few as one (e.g. methionine, tryptophan). • When bases in the anticodonare modified, further pairing patterns become possible in addition to those predicted by the regular and wobble pairing

7. Inosine, which is often present in the first position of the anticodon, can pair with any one of three bases, U, C, and A. • In contrast, modification of uracil (U) to 2-thiouracil restricts pairing to A alone, because only one hydrogen bond can form with G. • Codon bias = the frequencies with which different codons are used vary significantly between different organisms and between proteins expressed at high or low levels within the same organism.

8. Example • Mammalian genes commonly use AGG and AGA codons for arginine, whereas these are very rarely used in Escherichia coli. • E. coli is a bacterium often used for expression of recombinant human proteins. • Correlating with this observation, in E. coli, the tRNAArgthat reads the infrequently used AGG and AGA codons for arginine is present only at very low levels.

9. Example 2 • Tetrahymena, the ciliate that played an important role in the discovery of telomerase, possesses tRNAs that read the canonical stop codons UAA and UAG as glutamine (Gln), making these genes impossible to express heterologously without some type of redesign strategy of the gene or host.

11. Peptide Bonds

12. Four Levels of Structure

13. Globular proteins • The overall shape of most proteins is roughly spherical. • Proteins that adopt this form are called globular proteins. • E.glysozymefolds up into a globular tertiary structure, forming the active site within a deep pocket between folded regions.

14. Fibrous proteins • Fibrous proteins have properties distinct from globular proteins. • A common feature of most fibrous proteins is their long filamentous or “rod-like” structure. • A triple helical arrangement of polypeptide chains is exemplified by the collagen family of proteins, which are a major structural component of skin, tendons, ligaments, teeth, and bone. • The α-keratins, which are structural components of mammalian hooves, nails, and hair, adopt a structure composed of “coiled coils” of α-helices. • A variation on this helical theme is structure of actinfilament.

16. Size and complexity of proteins • There is tremendous variation in the size and complexity of proteins. • The molecular weight of proteins and the number of subunits (polypeptide chains) shows much diversity. • Dalton units (1 Da is equivalent to 1 atomic mass unit) are used frequently in the protein literature to describe the molecular weight. • This is the absolute molecular weight representing the mass in grams of 1 mole of protein. • Typical polypeptide chains have molecular weights ranging from 20 to 70 kDa (20,000–70,000 Da).

17. Protein function • Proteins provide the structures that give cells integrity and shape – cytoskeleton. • Others serve as hormones to carry signals from one cell to another, or to transport oxygen around the bodies of multicellular organisms. • Of particular significance for molecular biologists are the proteins that mediate the activities of genes at all points in the flow of genetic information from replication to transcription to translation. • Another vital role of proteins is to serve as enzymes that catalyze the hundreds of chemical reactions necessary for life.

18. Regulation of protein activity by post-translational modifications • After translation, proteins are joined covalently and noncovalently with other molecules. • Complexes that form between lipids and proteins = lipoproteins, • Complexes that form between carbohydrates and proteins = glycoproteins • Complexes that form between metal ions and proteins = metalloproteins.

19. Post-translational modifications can have both structural and regulatory functions. • Important modifications include methylation, acetylation, ubiquitinylation, and sumoylation. • The most common regulatory reaction in molecular biology is the reversible phosphorylation of amino acid side chains. • Many steps in gene expression and cell signaling pathways involve posttranslational modification of proteins by phosphorylation. • Kinasescatalyze the addition of phosphate groups, whereas enzymes called phosphatases remove phosphates. • Kinasestend to be very specific, acting on a very few substrates. • Phosphatasestend to be nonspecific. • Many kinases self-regulate through autophosphorylation; many also can initiate reactions that are part of other negative feedback systems.

20. Two protein kinase groups have been widely studied in eukaryotes: • phosphorylatetyrosine side chains, • phosphorylateserine or threonine side chains. • Adding phosphate to a protein can cause it to change its shape, for example by masking or unmasking a catalytic domain; or the phosphorylated side chain itself can be part of a binding motif recognized by other proteins allowing proteins to dock and facilitating multiprotein complexes to form, or conversely to promote dissociation of a complex

21. Molecular chaperones • Molecular chaperones increase the efficiency of the overall process of protein folding by reducing the probability of competing reactions, such as aggregation. • ATP is required for most of the molecular chaperones to function with full efficiency. • Molecular chaperones include heat shock proteins, such as Hsp40, Hsp70, and Hsp90, which promote protein folding and aid in the destruction of misfoldedproteins • The designation of these as heat shock proteins reflects the fact that their concentrations are substantially increased during cellular stress.

23. Ubiquitin-mediated protein degradation • Cells have several intracellular proteolytic pathways for degrading: a) normal proteins whose concentration must be rapidly decreased, b) misfoldedor denatured proteins, c) foreign proteins taken up by a cell

25. Protein misfolding diseases