Proteins and Nucleic Acids

1. Proteins and Nucleic Acids Also featuring amino acids and polypeptides…..

2. Nucleic Acids • DNA and RNA are informational molecules in eukaryotic cells. • They are built of nucleotides (the purines guanine and adenine and the pyrimidines cytosine, thymine and uracil) with a sugar-phosphate backbone. • DNA has C-G and A-T; RNA has C-G and A-U.

3. Nucleic Acids

4. Polymerization to form the nucleic acid chain (As with other polymers, the linkage reaction involves hydrolysis. In this case a phosphodiester bond is formed.)

5. Crosslinks in the complementary pairing between purines and pyrimidines Hydrogen bonds stabilize the pairs of nucleotides in the DNA double helix.

6. Transcription: DNA ->RNA • 1. Binding: RNA polymerase binding to a DNA promoter sequence triggers localized unwinding of the double helix. • 2. RNA polymerase initiates synthesis of messenger RNA from one of the two DNA strands. • 3. Elongation of the RNA from complementary nucleotides occurs as the RNA polymerase moves along the DNA. • 4. RNA polymerase reads a termination sequence and causes the completed mRNA strand to dissociate from its DNA template.

7. DNA transcription

8. Polytene chromosomes reveal DNA regions that are undergoing transcription in a dipteran insect. Highly condensed DNA, known as heterochromatin: Arrows indicate the condensed X chromosome (Barr body) at the periphery of the nucleus of a female mammal.

9. Posttranscriptional Processing • After a primary transcript mRNA has been completed, • Introns – segments of the primary transcript that will not contribute to the protein sequence – are clipped out. • The remaining exons are spliced together to form the mature mRNA.

10. Posttranscriptional processing of the mRNA for the beta chain of hemoglobin

11. Splice variants • In many cases, the exons can be cut and spliced in a number of different ways – leading to splice variants. Splice variation is one way in which a single gene can give rise to multiple distinct proteins. Gene splicing is observed in high proportion of genes. In human cells, about 40-60% of the genes are known to exhibit alternative splicing.

12. Mechanisms that cause splice variants • There are several types of common gene splicing events. • Exon Skipping: This is the most common known gene splicing mechanism in which exon(s) are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. The exons are the coding regions of a gene and are responsible for producing proteins that are utilized in various cell types for a number of functions. • Intron Retention: An event in which all or part of an intron is retained in the final transcript. In humans 2-5 % of the genes have been reported to retain complete introns and about 95% retain small parts of introns. • Alternative 3' splice site and 5' splice site: Alternative gene splicing includes joining of different 5' and 3' splice site. In this kind of gene splicing, two or more alternative 5' splice site compete for joining to two or more alternate 3' splice site. • The consequences of alternative splicing may be trivial, functional or pathological.

14. It’s time to shift from nucleic acids to amino acids, peptides and proteins…

15. The relationship between nucleic acids and proteins was forged over a period of time in the dim past, and recent appreciation that some mitochondria, protoza and the Archaea differ from multicellular animal cells in details of the triplet code amino acid specification attest to splits in the ancestry during the period of “experimentation”.

16. Why only 20 amino acids? • There are at least 70 different amino acids, most of which are not found in proteins. • At some very early state of evolution of life, a commitment to use only L amino acids was made. This cuts the number of possibilities by half. • Some of the “forbidden” L amino acids are toxic.

17. The nature of amino acids • The building blocks of peptides and proteins can be divided into four categories, based on how they will interact within the protein structure/aqueous medium. • Nonpolar (10) : do not interact with water- located in interior of soluble proteins, or on exterior of intramembrane domains of membrane proteins • Polar (5) : hydrophilic, located in protein exterior for soluble proteins, or in interior of intramembrane domains of membrane proteins • Basic (3) : hydrophilic, form hydrogen bonds with water • Acidic (2) : very hydrophilic, usually located on the protein surface

18. Amino acids

19. A different text gives a different breakdown: 9 nonpolar, 6 polar, etc. It is cysteine that seems to not know where to go... .

20. Amino Acid Functions • Singaling molecules e.g. glycine, GABA (a glutamine derivative) and dopamine (a tyrosine derivative) are neurotransmitters • Metabolizable for energy • Sources of amine group in synthesis • Peptide and protein subunits

21. Peptides Small chains of amino acids (fewer than 40 amino acids – the smallest is 3 amino acids) serve as peptide hormones and neurotransmitters. Examples : • Oxytocin 9 amino acids CYIQNCPLG (C's are disulfide bonded). Uterine contraction, causes milk ejection in lactating females, responds to suckling reflex and estradiol, lowers steroid synthesis in testes • Vasopressin antidiuretic hormone, ADH) 9 amino acids CYFQNCPRG (C's are disulfide bonded) Responds to osmoreceptor which senses extracellular [Na+], blood pressure regulation, increases H2O readsorption from distal tubules in kidney • Melanocyte-stimulating hormones (MSH): a peptide = 13 amino acids, b polypeptide = 18 amino acids, g polypeptide = 12 amino acids. Pigmentation

22. Polypeptides are longer strings of amino acids (typically between 100 and 1000 peptide residues) • Amino acids are joined by peptide bonds between the alpha amino acid group (N terminus group) of one amino acid and the alpha carboxyl group (C terminus group) of the next amino acid. • Peptide bonds are formed in a dehydration reaction. • The sequence of amino acids is listed from the amino to the carboxyl end:

23. Translation: Three steps in protein synthesis 1. messenger RNAs (mRNAs) code for the amino acids of a polypeptide based on a triplet code. There are 3 stop codons and one initiation codon. (Note that there is not a 1:1 match of triplets and amino acids – there are 64 triplets and only 20 amino acids, so the code is redundant – some amino acids are coded for by multiple triplets.)

24. Translation, con’t. 2. Transfer RNAs (tRNAs) recognize amino acids (actually, a specific enzyme is required to make the attachment) and bring them to ribosomes, where they line them up based on their complementarity to the mRNA: GUG with CAC, etc.

25. Translation, con’t. • 3. mRNA is translated into polypeptides on ribosomes, which are composed of ribosomal RNA (rRNA) and ribosomal proteins.

26. Protein Structure: Four levels of organization

27. Chaperones: Protein folding is generally not spontaneous • While the sequence of nucleotides in DNA is being translated into a sequence of amino acids to form a protein, the charged groups in the chain of amino acids are interacting, folding to allow + to meet –, or hydrophobic groups to cling together to avoid water. Some proteins assume their tertiary shape spontaneously, but in others, this process is assisted by chaperones, proteins that temporarily stabilize the incomplete protein by blocking associations that would interfere with the bending pattern characteristic of the functional protein.

28. Diseases related to protein misfolding, aggregation and precipitation • Alzheimer’s Disease: Plaques of β amyloid result from aggregation and precipitation of partially folded 40-residue protein fragments; the presence of a misfolded fragment initiates aggregation of similar fragments. • Bovine spongioform encephalopathy (mad cow disease) and the related scrapie in sheep and Creutzfeldt-Jakob in humans are caused by prions. The prion protein normally plays a function in synapse modification in learning, but the abnormally folded proteins form insoluble fibrous aggregates. Prion protein Normal Diseased

29. Protein misfolding occurs frequently in normal cells • Chaperones don’t always prevent misfolding • Misfolded proteins are labeled for destruction by mechanisms that are involved in turnover of appropriately folded proteins (i.e. proteasomes and lysosomes, which you will hear about in a subsequent lecture)

30. Protein Functions • Enzymes – Catalysts • Regulation – transcription factors, protein hormones • Transport – hemoglobin for O2, membrane transport proteins • Storage – Ferritin can carry 4,500 iron molecules • Contraction – actin, myosin, tubulin • Structural – keratin, collagen • Protective – blood clotting, IGG’s

31. Posttranslational Processing • In posttranslational processing, parts of the protein structure are removed. It may be as simple as removal of the signalsequence that directs a protein to be secreted, or as complex as what happens to proopiomelanocortin (POMC) to yield adrenocorticotrophic hormone (ACTH) (shown in the next slide). • Like posttranscriptional processing, posttranslational processing enables one gene to have multiple products, violating the one gene – one protein rule that used to be a central dogma of molecular biology.

32. Post-translational processing of proopiomelanocortin (POMC). POMC in mammals consists of 3 exons, of which exons 2 and 3 are translated. Prohormone convertases 1 and 2 (PC1/2) break the parent POMC peptide into successively smaller peptides by cleavage at paired dibasic amino acid residues consisting of lysine (K) and/or arginine (R). The final products are generated in a tissue specific manner, for example α-MSH and ACTH are not produced by the same cells in the pituitary. The final products include the melanocortins (MSHs and ACTH), β-endorphin (β-end) and corticotrophin-like intermediate peptide (CLIP). There are intermediate peptides whose biological function remains unclear, such as β and γ lipotrophins (β-LPH, γ-LPH). Millington, Nutrition & Metabolism 2007, 4:18.

33. POMC is a single gene product that regulates many body activities • Depending on the type of neuron that is expressing it, POMC is converted to different endproducts that regulate a wide variety of activities, including feeding, stress responses, metabolism, pain perception, body pigmentation, sexual behavior, lactation, etc.

34. Insulin is another example of a protein that is created by postranslational processing In this process, the immature protein is secreted into the endoplasmic reticulum as it is synthesized. Within the ER, the signal sequence is then removed. The segments of the original gene product that will become the alpha and beta chains of the final hormone are folded by a chaperone protein and inked by disulfide bonds to form proinsulin. Removal of the C-peptide loop connecting the two chains results in the final hormone.

35. Summary Nucleic acids store information (DNA) and transfer this information into proteins (mRNA, tRNA, rRNA) Proteins are the most diverse class of molecules in cells; their roles are to execute the information carried in the DNA Protein function depends on protein structure. The 1o structure of a protein is established by posttranscriptional and posttranslational processing, and provides the basis for higher structural orders, which generally cannot be attained without the assistance of other proteins.