Advanced Molecular Biology

Molecular biology

Macromolecules and interactions

Macromolecules: high molecular weight composed by simple structural units, linked together by covalent bond. Identical or very similar, repeated many times and linear or branched. They are: proteins, nucleic acids, carbohydrates and lipids.

DNA

• History
  • 1950: Chargaff rules.
    • (T+C) = (A+G).
    • T = A, C = G.
    • A+T not always = C + G.
  • 1952: Helical form of the DNA by Franklin and Wilkins.
  • 1953: DNA structure published by Watson and Crick on Nature.

Structure: It’s a linear polymer of nucleotides, molecules consisting of:

• Deoxyribose: a pentose sugar. The presence of an H in position 2 confers stability.
• Nitrogen base: there are two types:
  • Purines: double ring, they are adenine and guanine.
  • Pyrimidines: single ring, they are cytosine, thymine.
• Phosphate group: one or more can bind to the nucleoside in position 5’ or 3’, forming the nucleotide and giving acid value. The nucleotides used as a synthesis substrate have 3 phosphate groups (α, β, γ).

The forces establishing the DNA secondary structure:

• H bonds among paired bases: 3 between G and C and 2 between A and T.
• Bases stacking: electrons move around the ring and the electron cloud is shared between stacked bases with hydrophobic interactions, it helps in the stability.
• Repulsions between P: the distance between the two negative charges is 2 nm, too short to ignore the repulsion and it’s against the stability. To lower there are histones in eukaryotes + ions inside cells like Na⁺, K⁺, Mg²⁺ in both eukaryotes and prokaryotes.

Alternative structures:

• DNA B: double helix in solution, very humid. It’s the most abundant form in cells: 0.34 nm the space between two nucleotides on the same strand and 3.4 nm the length of a complete turn (10 nucleotides).
• DNA A: dehydrated form of DNA, right-handed, bases stacking with major angle. Propeller pitch of 25 Å, diameter of 23 Å and 11 bp per helix turn. Also possible for RNA.
• DNA Z: left-hand, pitch of 46 Å and diameter of 18 Å. Generated by the change of orientation of the glycosidic bond from anti to syn in some cases. It exists in nature because specific proteins bind to it and can be found in a stretch of DNA B. Antibodies exist against this DNA.

Denaturation

The best method to quantify the amount of DNA is the denaturation of DNA using the Nanodrop (spectrophotometry: testing the light absorbance at the wavelength of 260 nm, property due to the aromatic rings of the bases of DNA). Opening the double helix there’s a hyper-chromatic shift: the same fragment of DNA absorbs light at 260 nm in different ways when it’s in a double helix and in single helices (hyper → a bigger amount of light is absorbed → shift). By heating, energy is given to the molecule, the movement of the atoms inside the structure increases and weak bonds such as H-bonds are broken, so the DNA is separated. Bases with 2 H-bonds (A-T) denature before than bases with 3 H-bonds (G-C), so denaturation starts with bubbles opening in the molecule and at last we have a fully denatured DNA.

T_m = melting temperature, at which half of the molecules of DNA is in a denatured form and half is still in a double helix. It depends on the number of H-bonds present, so on the content of A-T and G-C bases and it’s characteristic of different DNA molecules. A poli-AT has a lower T_m than a poli-GC; normally DNA molecules have a T_m between the two extremes.

Is it possible to change the T_m of a particular molecule of DNA? Yes, there are many ways other than heating:

• By changing the pH (by increasing the pH, in basic conditions the OH^- can accept H-bonds and can compete with the base paired lowering the T_m).
• Another possibility is removing positive ions which stabilize the DNA molecule.
• By adding urea or formamide (denaturing agents), they enter the double helix and brake H-bonds because they are competing.
• By affecting the hydrophobic interactions: placing something hydrophobic to give distortion to the molecule like organic solvents (e.g., DMSO).

RNA

Structure: It’s a linear polymer of nucleotides, molecules consisting of:

• Ribose: pentose sugar with in the presence of a –OH in position 2, which confers instability.
• Nitrogen base: there are two types:
  • Purines: double ring, they are adenine and guanine.
  • Pyrimidines: single ring, they are cytosine and uracil.
• Phosphate group: one or more can bind to the nucleoside in position 5’ or 3’, forming the nucleotide and giving acid value. The nucleotides used as a synthesis substrate have 3 phosphate groups (α, β, γ).

RNA can form complicated 3D structures where the strands can loop back and form intra-strand base-pairs from self-complementary regions along the chain, like rRNA and tRNA.

mRNA messenger

In the 5’-end in prokaryotes mRNA the most important sequence is the RBS (Shine-Dalgarno), while in eukaryotes the recognition of RBS is more difficult.

• Prokaryotic Bacterial mRNA is polycistronic. Even in bacterial cells there are regions of RNA that can acquire particular structures: terminator and anti-terminator in prokaryotic RNA.
• It’s the way RNA polymerase in prokaryotes stops transcription. In particular operons, mRNA contains region to stop transcription, but before there is an extended top structure. The part in yellow can pair with the pink one: we can have 2 alternative forms A and B. The features of the termination are no more present in B: anti-terminator (avoid the termination of the transcription - Trp operon).
• Eukaryotic 5’UTR and 3’UTR, in between there is the coding sequence. In 5’ and 3’ UTR there are a lot of informations and secondary structures that RNA can assume, essential for the function of that RNA.

An hairpin can avoid the translation of a particular mRNA. Or a stem loop can be the binding site of a protein, like one that blocks the mRNA transcription. An IRES sequence is an Internal Ribosomal Entry Site: interesting sequence for biotechnologies, ribosomes can bind to this sequence to start the translation of that mRNA. This is important when the mRNA doesn’t present a CAP in the very beginning, this usually means no translation. Create an expression vector in which the expression of two proteins can be induced starting from the same promoter, the first coding sequence can be translated using the CAP and the second using the IRES (just before this coding sequence). Use this technique to produce a protein composed by two subunits using just one plasmid, because using 2 plasmids there’s ratio issue when they enter the cells. At the 3’ UTR there is the poly-A tale, essential to terminate the transcription and for the stability of mRNA. In this region there are others parts like: sequence that helps an additional poly-adenylation when the mRNA is in the cytosol, binding site for proteins that protect mRNA from endonuclease activity and regions where antisense RNA can bind.

mRNA in eukaryotic cells is always covered by a lot of proteins. The ones in the coding regions are the ones reminding the splicing reaction: usually, they help to identify the boundary between exons-introns. After the splicing, the protein remains on the RNA. These proteins are also the signal that tells to the nucleus that the RNA is mature and ready to be exported into the cytosol. Once the RNA leaves the nucleus several of these proteins are removed and replaced by cytosolic proteins (for stability, delivery, etcetera).

Eukaryotic small RNAs

pri-miRNA: is the primordial micro RNA, that assumes a particular form that ends with a loop and have 5’ and 3’ end free. snRNA U: a small nuclear RNA involved in splicing: it recognizes the boundary between eons and introns by base pairing and it is important to recognize the proteins important in the splicing reaction.

Proteins

Characterized by:

• Primary structure: aa sequence.
• Secondary structure: α-helix and β-sheet.
• Tertiary structure: motifs like zinc finger and helix-turn-helix (both bind grooves).
• Quaternary structure: domains. Present just in some proteins like hemoglobin.

Allosteric protein: it changes its structure as the consequence of ligand binding.

Distribution-sorting-secretion of proteins in cells: Signals are required. Gram- bacteria have 3 ways to secrete proteins:

• SEC Pathway: after protein synthesis there are chaperons that recognize them and allow their unfolding so they can use SecYEG translocon to go in the periplasm.
• SRP Pathway: ribosome attached to SecYEG translocon, so proteins are directly secreted in the periplasm.
• TAT Pathway: the protein immediately reaches the native conformation, very stable, so the TAT translocon is required to move the folded protein from cytoplasm to periplasm.

For Gram+ it’s easier: just one membrane, but still TAT and Sec translocons.

Macromolecule interactions

DNA – proteins: Specific interaction: recognition of a sequence of bases by aa. Unspecific interaction: independent from the base sequence. They are always weak interactions: H-bonds, hydrophobic, negatively-positively charged interactions. They must be weak to be reversible. The edge of each base pair is exposed in the major and minor grooves. It has a precise pattern of hydrogen acceptors and donors and hydrophobic groups that can be recognized by the aa of an interacting protein. In major groove, each of the 4 configurations presents a unique meaning, in the minor groove the 4 configurations present repetitive meaning. The recognizable code is particularly varied in the major groove where also more space is available. Most of the proteins bind DNA in the major groove, with few exceptions.

In the major groove of DNA there is more space. Specific interactions are favored in the major groove, the protein can fit better because there is more space. In addition, in the major groove is easier the recognition of specific bases: there is a lot of chemical information available for the interaction with protein residues. In the minor groove, there is less difference in the chemical information. A protein binds DNA because it recognizes a specific nucleotide sequence, the protein surface is “complementary” to the double-helix surface of DNA. Several sites of contact are necessary: each of them are weak, per se, however, as they are many (at least 20), the global binding force assures a specific and stable interaction. Proteins that bind DNA normally present more than 1 subunit to increase the affinity for the binding. By looking at the structure of proteins, like transcription factors, this feature is clear. Their consensus site on DNA is accordingly symmetric (palindromic sequences). TFs very often act as dimer, tetramer and so on, rarely act as a single molecule. Usually, α-helices bind DNA. Exception: TBP binding the TATA box. In this case the interaction occurs in the minor groove of the DNA and through β-sheet structures. The interaction is quite strange because it’s possible but it leads to a distortion of the double helix of DNA, bending of about 80°. Plus, the protein is a monomer. The C-terminal of the protein has been duplicated during evolution and even if it’s still a monomer this contributes to the interaction with DNA. Another example: interactions of DNA with histones are unspecific and are mostly with proteins with a positive charge (since DNA has a negative charge). With the formation of nucleosomes, the contact sites are mainly in the minor groove but especially on the backbone (with the P). There are about 40 H-bonds among basic aa and DNA phosphates and bases in the minor groove. To remove nucleosomes or to shift them ATP is to break the interactions.

RNA – proteins: RNA can interact with proteins. RNA is a single strand so the protein can interact directly with the bases. In other cases, the RNA can assume the A double helix conformation and in this case, there are the same interactions described for the DNA. The recognition motifs of proteins for DNA are few and usually involve an α-helix. For RNA, there is a lot of different motifs because the bases sequence can be very different.

Protein – protein: Interactions occur between two complementary surfaces (e.g., antigen and antibody). In eukaryotic cells there are a lot of compartments but proteins are synthesized for the majority in the cytosol so they have to reach all the different compartments. They have to be recognized thanks to signals by molecules that deliver them to the right compartment or for the secretion.

Gene structure and transcription

Gene: segment of DNA, containing an information. It’s the basic physical and functional unit of heredity. It’s a specific sequence of nucleotides in DNA or RNA. It’s usually located on a chromosome. It controls transmission and expression of one or more traits. It can specify a product (protein or RNA) that controls the function of other genetic material.

Structure

The structure must:

• Be constant, in order to transfer the information between generations without changes (semiconservative DNA replication).
• Contain the instruction for the regulated production of RNA (gene expression).
• Evolve (mutations and selection).

Prokaryotes: Very often genes involved in the same metabolic pathway are clustered in operons. There is the transcribed region, made up of different genes, and upstream there is the control region, the promoter -10 and -35, where RNA polymerase can bind (binds at -35 and start melting DNA at -10) + the operator region to regulate transcription, together with regulatory proteins. Additional control elements can improve the transcription rate such as enhancers and UP elements, that are recognized by the α-subunit of the RNA pol, so this element is quite near to the promoter. There is also the terminator sequence that tells the pol to stop the transcription. The product of transcription is a single polycistronic RNA containing the different coding regions of the genes, each presenting the starting point and the STOP codon for translation. Between coding regions, there is the RBS (ribosome binding site) to start the translation. The product does not need processing.

Eukaryotes: There are single genes that code one molecule and in most cases genes are interrupted by introns. So there are coding and non-coding regions. Some genes coding for microRNAs codes for a single transcript that can be processed to give birth to different products: different microRNAs. Usually, there’s a transcribed region made of exons + introns and the control region. The control region can be divided in a proximal control region, containing the real promoter, located across the starting point (-50 + 30 more or less), and a distal control region. In many promoters there is the TATA box for the binding of the TBP. The TBP is always present in eukaryotes at any given time, but when the TATA box is not present the TBP can stay in the promoter region thanks to the interaction with other proteins that can bind the DNA with indirect interaction. In eukaryotes, enhancers are very important and can be close or very far from the promoter region. They can also be internal to the coding sequence, for example in an intron. Response elements: DNA sequences recognized by specific transcription factors (TFs). They are many within the control regions and they also can be recurrent in different regions. The regulation of the expression is mediated by different TFs and there’s a combinatorial organization of these responsive elements. For the transcription of a single gene, all the sequences must be recognized by the specific TFs. In RNA pol II there is the CTD (C-terminal tail), important for the initiation of gene transcription. A single primary transcript is obtained and has to be processed with 5’ capping, splicing, and 3’ polyadenylation.

Transcription

In eukaryotes there are 3 specialized RNA pol, in prokaryotes just one. Eukaryotic RNA pols are the evolution of the prokaryotic one.

• Core enzyme of RNA prokaryotic pol, responsible for the catalytic process, is β’ + β + α₁ + α₂ + ω. This is unable to start the transcription at the right place, because it’s missing the σ factor, an additional subunit that leads the interaction with the promoter region.
• For eukaryotes, there are other subunits forming the core, plus the subunits common to all RNA pol types. So there are additional enzyme-specific subunits that allow the pol to work better on specific genes, giving specificity:
  • RNA pol I: large rRNA precursor.
  • RNA pol II: mRNA, microRNA.
  • RNA pol III: tRNA.

In prokaryotic cells, the σ factor recognizes the promoter and induces the melting of the DNA. It transforms the core enzyme in a specific protein. At the end, σ factor must leave the RNA pol to allow the movement through the DNA. In eukaryotic cells, there’s a more complex situation. The RNA pol is a sort of a core, unable to bind to the promoter. A large number of general TFs are required to recognize the promoter, they act like σ factor in a certain way. The transcription starts just when the CTD is modified by phosphorylation. Then, there is the melting of the DNA by TFIIH with helicase activity, that also acts as a kinase to phosphorylate the CTD of the protein. TFIIH will move together with the pol during the transcription.

• Termination region folds into a loop to stop transcription: intrinsic termination, in prokaryotes.
• In eukaryotes is different because the mRNA have to be processed in three ways. The 5’ capping occurs immediately after transcription. The splicing reaction starts during transcription. The Poly- A site is recognized by specific enzymes and the polyadenylation occurs. The different steps are regulated by different types of phosphorylation of the tail of RNA polymerase II. Once the RNA is mature it can leave the nucleus.
• In prokaryotes transcription and translation occur both in cytoplasm.