Saras powerpointsvar

From Carls wiki

Här kommer ett bidrag från mig. Jag har samlat ihop svaren från alla powerpointpresentationer så gott jag kunnat utan att behöva förstå allt för mycket. Hoppas att nåt kan vara till hjälp, trots alla oklarheter.

/Sara

1 Transcription
2 Protein Structure
3 Alpha, beta and alpha&beta proteins
4 Transcription regulation in prokaryotes
5 Transcription regulation in eukaryotes
6 Non-coding RNAs
7 Viruses
8 Talal's lectures about signal transduction
9 Jan's lectures about prokaryotic genomes
10 Microtubules

Transcription

1. What is initiation, elongation and termination? Describe the addition of a single nucleotide (insertion, covalent bond formation and translocation).

Initiation: Polymerase binds to a promoter sequence in duplex DNA (closed complex). Polymerase melts the duplex DNA near the transcription start site, forming a transcription bubble (open complex). Polymerase catalyses the phosphodiester linkage between the two initial rNTPs.
Elongation: Polymerase advances 3’ – 5’ down the template strand, melting duplex DNA and adding rNTPs to the growing DNA.
Termination: At the transcription stop site, polymerase releases the completed RNA and dissociates from DNA.

Substrate complex(insertion) → pre-translocation (product) complex → (Translocation – pyrophosphate) Post-translocation complex.

2. What are the similar and different features of T7 RNA pol and eukaryotic RNA pol II?

Translocation: T7 has a translocation, after release of pyrophosphate the O helix rotates which moves the RNA and also Tyr639 and results in a translocation. RNA pol II has a bridge helix that changes the conformation from straight to bent resulting in a translocation similar to T7. However, the helix interacts with DNA, not RNA as in T7 and there is no Tyr involved. RNA pol II has jaws to position the incoming DNA strands.
Catalytic activity: Both T7 and pol II have two magnesium ions involved.
Sequence: No sequence similarities between T7 and pol II.
Nucleotide addition: In pol II nucleotide addition is a two step process requiring a rotation of a base. There is also no obvious sugar selection, like Tyr639 in T7. The sugar selection is probably carried out during the rotation of the base.

3. What eukaryotic promoter elements do you know? Draw a schematic picture! (you do not have to know the all sequences...)

The TATA-box, the most abundant promoter.
CpG-islands: CG rich stretches of 20-50 nucleotides within 100 base pairs upstream of the start site.
Inr: Initiator. Some promoters contain both initiator and TATA-box.
BRE sequence: located immediately upstream of the TATA element and increases the affinity of TFIIB for the promoter.
DPE: Downstream promoter elements, 30 nts downstream of the transcription site.

4. TFIID and its functions. How does TBP bend the DNA?

Transcription Factor IID is composed of 14 subunits, one of them being the TATA box binding protein, TBP. It functions as a promoter recogniser. The TBP box makes a pseudo-twofold sequence-specific interaction with two Thr and twp Asp of the TATA box. The TBP is responsible for stacking between base and Phe.

5. TFIIB and its functions. Which interactions determine "rough" and "fine" positioning of RNA PolII on the transcription start site?

TFIIB functions as the start site selection for Pol II. Some TFIIB mutants result in a shift of transcription start site. Its CTD interacts with TBP and the DNA around the promoter. N-terminal domain interacts with Pol II.

Rough positioning is determined by interaction of TFIIB CTD with TBP. Fine positioning is due to interaction with DNA.

6. TFIIF and TFIIE and their functions.

TFIIF recruits Pol II to the existing DNA-TFIID-B complex. It positions Pol II over the start site and binds to the non-template DNA strand. It also induces non-specific binding of RNA pol II to DNA. It is a heterotetramer made from two subunits. Some regions are involved in transcription initiation and elongation.

TFIIE is a heterotetrameric protein. It creates a docking site for the next transcription factor: TFIIH. It also modulates TFIIH enzymatic activities. It also enhances promoter melting.

7. TFIIH and its functions.

TFIIH is a multimeric protein composed of nine subunits, some of them with distinct enzymatic activities.

It has a helicase activity, which unwinds DNA duplex at the start site, allowing Pol II to bind to the template strand. It has a kinase activity, which phosphorylates Pol II in the beginning of the elongation. Some subunits recruit DNA-repairing enzymes if the polymerase reaches a damaged region in the DNA.

8. TFIIA and TAFs.

TFIIA is required for transcription in vivo. Its function is a bit unclear, but it might help the other factors to bind. It has been shown to have some anti-repressor functions. It is not required for transcription in vitro.

TATA box binding protein associated factiors, TAFs, make up 13 of the subunits of TFIID. Some of them seem to be necessary for transcription initiation from promoters lacking TATA box. Other TAFs are tissue-specific coactivators. They also interact with other General Transcription Factors stabilising the complex. The 3D structure of some of them resemble histones.

9. What is Pol II pausing, backsliding and arrest?

Backsliding is an event when RNA polymerase moves backwards and newly made RNA gets inserted in the funnel. It can be caused by incorporation of the wrong NTP or when 3’ OH loses contact with the active site Mg(2+).

Pausing is when the backsliding RNA piece is 2-4 nts long and therefore is a reversible process so that the RNA polymerase can recover by itself.

Arrest is when the backsliding piece is longer, 7-10 nts, and gets trapped in a funnel pore so that the RNA polymerase cannot recover by itself. It can be overcome by special elongation factors which help RNA Pol II to cleave the arrested RNA.

10. What is transcription elongation factor TFIIS and how does it work?

Domain three of TFIIS gets inserted into the active site of Pol II. Domain two docks on the surface. Its acidic residues Asp290 and Glu291 assist in cleaving the phosphodiester bond allowing the backtracked RNA fragment to be released and RNA Pol II rescued from arrest.

Protein Structure

1. Amino acids and their properties. Hydrophobic, polar and charged amino acids. What is special about glycine, proline and cysteine?

Hydrophobic, aka polar, amino acids are

Polar amino acids are

Charged amino acids are:

Bases: His
Lys
Arg
Acids: Glu
Asp

Special amino acids are:

Glycine: doesn’t have a side chain. It consists of a NH2, a COOH and two Hs. Bad helix.
Cysteine: can form dimers with a disulphide bridge.
Proline: is circular, its N is bound to its side chain. Bad helix.

2. Omega angle, cis- and trans- peptides. Why cis- peptides are unusual? Why is it that most cis- peptides (when they do occur) are found in prolines?

Omega angle: between N and C in the peptide bond.
Psi angle: between C-alpha and C=O
Phi angle: between C-alpha and N-H.

Cis angles are energetically extremely unfavourable (1000 ×) because of steric clashes. In trans the omega angle is ~180o and 0 for cis. In proline the cis- peptide is only 4 times less favourable than the trans- peptide because there are steric clashes in both forms. Proline cis formation is important in protein folding. In most cases cis- peptides occur to maintain some particular conformation in the active site of the enzyme.

3. Which are phi and psi angles of the peptide backbone? What is a Ramachandran plot? Why amino acids in protein structures cluster in particular regions in Ramachandran plot? Why glycine can be found anywhere in Ramachandran plot?

The peptide backbone in a right handed alpha helix form has

(φ, ψ) = (-30 — -180, -10 — -50)

A beta sheet is found at angles

(φ, ψ) = (-30 — -180, 10 — 180)

A Ramachandran plot is a plot of ψ (psi) against φ (phi). Only certain combinations of angles are allowed. Glycine lacks the C-beta and can therefore occur anywhere in the Ramachandran plot.

4. What is a hydrophobic core of protein? What kind of amino acids are located in the core? What kinds of amino acids are normally located on the surface of protein?

The hydrophobic side chains of a protein tend to cluster together to avoid any unnecessary contacts with water molecules. In general, the hydrophobic core is located in the interior of the protein, forming a hydrophobic core.

Polar and charged amino acids are usually found at the surface of the protein. They are seldom buried within the core, and when they are they are almost always involved in hydrogen bond formation.

5. What are alpha helices? What is the difference between alpha helix, π helix and 3₁₀ helix? Do alpha helices usually have right or left hand?

alpha helices: a secondary structure of peptides. They have 3.6 residues per turn. There are hydrogen bonds between every fourth residue (regular alpha). They are always right-handed.
3₁₀ helix: has hydrogen bonds between every third residue and has 3 residues per turn. It is very compact, the atoms are packed tightly.
π helix: has a hole in the middle of the helix. There are h bonds between every five residues.

The latter two helices are energetically unfavourable and very rare. They only occur at the ends of alpha helices or in separate single turn helices.

There are a few cases of left-handed helices, but they only occur to give special functions to e.g. a ligand binding or an active site.

6. What are beta strands and beta sheets? What is the difference between paralel and antiparalel beta strands?

Beta strands are the other secondary structure. They are straight but pleated. Beta sheets are formed from several strands that are bonded to each other with hydrogen bonds. They can be either parallel or anti-parallel. If the strands follow the same direction (N-Ca-C-N) the sheets will be parallel. If not they are anti-parallel.

7. What are protein motifs? Tell about helix-turn-helix, beta hairpin, helix-strand-helix and greek key motifs!

Protein motifs are simple combinations of a few secondary structure elements that occur frequently in protein structures. These units are called supersecondary structures or motifs. Some of them can be associated with specific biological functions, e.g. DNA binding. Others don’t have any special biological function alone, but are part of larger structural and functional assemblies.

helix-loop-helix motif: consists of two alpha helices joined by a loop. It’s a DNA binding motif.
beta hairpin motif: consists of two adjacent antiparallel beta strands joined by a loop. It can occur both as an isolated unit or as a bigger beta sheet.
Greek key motif: the most common way to connect four adjacent antiparallel beta strands.
beta-alpha-beta motif: is a convenient way to connect two parallel beta strands. You have two beta strands connected with an alpha helix. The helix can be either below or above the plane of the beta sheet.

8. What are protein domains?

Domains are polypeptide chains or part of chains that can fold independently in a stable tertiary structure with its own hydrophobic core. They can be formed from several simple motifs and additional secondary structure elements. In proteins with several domains, most often, each domain is associated with a distinct biological function.

There are also a few cases where it takes more than one protein to form a single domain.

Alpha, beta and alpha&beta proteins

1. Describe coiled-coil alpha helical structure!

Two alpha helices wound together form a coil-coil alpha helical structure. This way their hydrophobic core gets packed together and they become more stable. Since it takes ~3,5 residues per turn which means that every seven residue is repeated – heptad repeat. Residues A and D are hydrophobic and E and G are polar contributing to salt bridges between the helices for stabilisation and orientation.

2. Describe four helix bundle structure!

Four helices arranged in a bundle with the helical axes almost parallel to each other can form a complete domain. There is a hydrophobic core at the center of the bundle and hydrophilic residues at the surface. It occurs in many proteins. Usually the helices are antiparallel. There is one form where the bundle is made of two pairs of parallel alpha helices joined in an antiparallel fashion.

3. In which two ways can two alpha helices pack each against other? Describe them!

alpha helices: can pack against each other in either ridges in grooves or knobs in holes.
ridges in grooves: the side chains in an alpha helix are arragned in a helical row along the surface of the helix. This leads to the formation of ridges spearated by shallow grooves on the surface. The ridges and grooves that are formed are usually 3-4 aa apart.
knobs in holes: need a heptad repeat. Each side chain in the hydrophobic residue from one helix can contact four side chains from the second helix. The helices are arranged such that the two d-residues face each other.

4. What is a globin fold? What is hemoglobin? What is the molecular basis for sickle-cell anemia?

The globin fold is one of the most important folds. It is found preserved in many unrelated proteins. There are 8 different alpha helices packed around the core such that the sequence consecutive helices are not adjacent in space except for helices G and H which are antiparallel.

It is preserved through evolution because the hydrophobic core is preserved and the helical movements accomodate new mutations.

Hemoglobin is a tetramer built up of two copies each of different peptide chains alpha and beta globin chain in normal adults. Each chain has the globin fold with a haeme pocket.

Sickle-cell anemia is caused by a mutation of Glu6 into a Val which produces a patch on the surface. The patch happends to fit and bind a hydrophobic pocket in the deoxygenated form of another hemoglobin tetramer. Due to the high concentration this leads to polymerisation of the hemoglobin molecules into fibres, deforming the erythrocite into a sickle shape. However, the disease gives protection against malaria and has been preserved. It is not a lethal mutation in heterozygots, only in homozygots.

5. What kinds of beta barrels you know? What is the difference among them?

Antiparallel stranded barrels:

up & down barrel: has the simplest topology: the strands are antiparallel and connected by hairpins.
greek-key motif: can also be a type of barrel. It crosses on the side. It goes up and down.
Jelly roll barrel: always has the up and down structure and forms a perfect barrel by crossing over and below. This barrel has four greek motif connections.

Parallel stranded barrels:

TIM barrel: consists of twisted parallel beta strands arranged like the staves of a barrel, with alpha helices in between.
Rossman fold/Open twisted barrel: an open twisted beta sheet surrounded by alpha helices on both sides. The plane has alpha helices both above and below.

6. What are the beta propeller proteins? Where is the active site usually located in them?

The beta propeller is antiparallel and has high repetition. It is grouped four by four in a flower shape. The active site is found on the edge of the opening, typ på toppen av tältet.

The loop regions that connect the motifs and the loops that connect strand 2 and 3 form a wide funnel shaped pocket containing the active site.

7. What are beta helical proteins? How do two and three sheet beta helices look like?

The beta helix is formed by beta strands separated by loop regions. The polypeptide chain is folded into a wide helix with 2 or 3 beta strands for each turn. The strands align to form either 2 or 3 parallel beta sheets with a core between the sheets completely filled with side chains.

The two sheet beta helix is held together or stabilised by Calcium ions. The sheets are separated in levels with two in each. There are 18 residues, 3 in each beta strand and 6 in each loop.

The three sheet beta helix has three short strands with 3 to 5 residues. Three loop regions connect the strands. Loop a is fixed to 2 residues while the other two vary in length. It is remarkably stable. The number of helical turn in this motif is greater than in the two sheet beta helix. For example there can be 7 turns.

8. What are TIM barrels? Where is the active site usually located?

TIM barrels are usually composed of 8 parallel beta sheets with sheet 8 connecting to sheet 1. It is one of the largest and most easily recognisable motifs. It is found in many unrelated enzymes. A minimum of 200 amino acids are required. 160 of these are conserved and form the secondary structure. Loops contain the catalytic amino acids. Between the twisted parallel sheets there is a hydrophobic core. The hydrophobic side chains of the alpha helices pack against the hydrophobic side chains of the beta sheets.

The active site is usually found at the bottom of the funnel shaped pocket created by the 8 looks that connect the CTD of the beta strands with the N end of the alpha helices.

9. What are open α/β sheet structures? Where is the active site in them?

Open alpha beta structure has alpha helices on both sides of the beta sheet. There are always two strands in the interior of the sheet whose connections to the flanking strand are on opposite sides. E.g. Rossman fold belongs to this superstructure. There can be a variation if topology with 4-10 beta strands and mixed beta sheets.

There are two hydrophobic cores created between the alpha helices and beta strands on each side of the central sheet. A crevice is formed in this interface and that is where the active site is found.

10. What is a horseshoe fold? Where is the hydrophobic core in those structures?

This fold is usually Leu rich. It is an all parallel beta sheet that is open. All alpha helices are found on one side of the beta sheet. Only one side of the beta sheet is exposed to the solvent. The hydrophobic core is found at the interface between the alpha and beta.

Transcription regulation in prokaryotes

1. Can you describe, how helix-turn-helix DNA binding proteins interact with DNA? How does activation/inactivation of the dimeric helix-turn-helix proteins work?

Helix-turn-helix is the most common DNA binding motif in prokaryotes. It is present in most transcription repressors and activators. One of the helices, the DNA recognition helix, gets inserted in the major groove of DNA. The major groove differentiates between all four nucleotides, while the minor groove only differentiates between base pairs (AT, CG).

Helix-turn-helix proteins are often dimeric, with two recognition helices recognising two adjacent DNA sequences. It is dimeric because a dimer binds stronger to DNA than does a monomer. By changing the relative positions of the monomers, the dimer activity can be turned on and off. A ligand changes the position of DNA binding to alpha helices so that they don’t bind to DNA anymore or the ligand changes the position of the helices so that they do bind to DNA.

The hydrogen bonds between the sugar-phosphate backbone and the protein help to anchor the protein to DNA. Sequence-specific interactions between DNA and the recognition helix determine what regions of the DNA that will interact. Sequence non-specific interactions contribute to the overall stability but don’t differentiate the bound DNA sequence.

2. What prokaryotic promoter elements do you know? What are the sigma factors?

Bacterial promoters have -35 and -10 elements. Some have UP elements (AT-rich motifs found >-35 from the transcription start site that stimulate the basal transcription from the promoter). There are also extended -10 elements in some promoters that lack -35 elements.

There are consitutive and inducible promoters. The first belong to genes that are transcribed at all times and circumstances. The latter belongs to genes that need to be transcribed only under certain circumstances or periods in the cell life cycle.

Sigma factors are required for promoter recognition and transcription initiation in prokaryotes. They have analogous functions like general transcription factors in eukaryotes. For expression from most promoters sigma70 is required. It is responsible for cell growth in all conditions, while other sigma factors are required for special events such as nitrogen regulation and sporulation.

3. Describe the tryptophan repressor mechanism!

The tryptophan repressor acts by steric hindrance. It controls the operon for the synthesis of L-tryptophan in E.coli by a simple negative feedback loop. In the absence of Trp the Trp repressor shows no affinity to the promoter and the RNA polymerase transcribes the operon. Instead, when enough Trp is made it binds to the repressor which now is able to bind to the promoter and block RNA polymerase from binding and transcribing.

4. How does lac repressor work?

The lac repressor is a homotetramer that works through steric hindrance. It binds both to the major and the minor groove of DNA. In the absence of lactose the lac repressor is bound to the promoter and blocks Pol-sigma70. When lactose is present it binds to the lac repressor which is released to let the polymerase bind to the promoter and transcribe the operon.

The functional lac repressor’s dimers each bind to a distinct DNA sequence at -82 and +11 from the transcription site. This results in DNA looping, preventing the DNA polymerase from binding to the -35 and -10 elements.

5. What is CAP an how does it work?

CAP is Catabolite Activator Protein. It activates transcription from more than 150 promoters in E.coli. Upon activation by cAMP (cyclic Adenosine MonoPhosphate) CAP binds to the promoter and helps RNAP-sigma to bind as well.

All CAP-dependent promoters have weak -35 sequences so that RNAP-sigma is unable to bind the promoter without CAP assistance.

CAP interacts with the CTD of the alpha subunit of RNAP, it recruits RNAP and helps position RNAP. It stimulates the basal transcription from the promoter.

There are three classes of CAPs. Class III requires two CAP dimers or one CAP dimer and one other regulation-specific activator. Class I interacts with the CTD of the alpha subunit of RNAP. Class II interacts with both CTD and NTD. Class III is similar to I and II except that each alpha CTD makes different interactions.

cAMP binds to the N-terminal domain of the DNA and causes two helices to reorient which moves the DNA binding domains apart and CAP can bind to DNA. This results in the DNA bending 80 degrees. CAP also activates the lac operon. When both cAMP and Pol-sigma70 bind to the promoter and the lac repressor is released the transcription of lacZ is high.

6. How do MerR family activators work?

MerR is an activator that controls genes involved in the response to Mercury poisoning. There are other MerR family activators that respond to a variety of different toxic compounds such as other heavy metals or drugs. In MerR activated promoters the -35 and -10 elements are separated by 19 bp instead of 17.

A member of the MerR family activators bind to a toxic compound. Both in the toxin bound and unbound form the MerR will bind to the promoter, but only the toxin bound form induces transcription. When the -35 and -10 elements are 19 bps apart they are both too far from each other but also on opposite sides of the helix, which prevents the promoter from binding. The DNA double helix is underwound by MerR so that the -35 and -10 elements are at the same distance as in normal spacing. This causes change in protein structure, but it is unclear how the drug binding to MerR induces underwinding of DNA.

7. What is rho-dependent and rho-independent termination? What is antitermination?

In prokaryotes there are two types of transciption termination: rho-dependent and rho-independent.

Rho-independent termination is acheived by a secondary structure of mRNA, an RNA stem-loop, followed by an AU-rich region. The elongation procedure is delayed by the folding of a hairpin which leads to an induced pausing. The next step is completion of the hairpin folding which results in dissociation.

Rho-dependent termination requires a rho protein. As polymerase transcribes away from the promoter, a rho factor binds to RNA and follows the polymerase. When polymerase reaches some sort of pause site, the rho factor catches up with the polymerase and unwinds the DNA-RNA hybrid, resulting in a release of the polymerase.

Transcription regulation in eukaryotes

1. Structural classification of eukaryotic transcription factor domains

Homeodomains: Sequences of 60 residues that function as DNA binding domains of transcription factors. They are built from three helices, where helix 2 and 3 form helix-turn-helix motifs similar to those in prokaryotic DNA binding proteins. They were first identified in Drosophila where mutant homeodomains cause legs growing from the head, etc. They can work in tandem with similar of different DNA binding domains.

Zinc fingers: A motif with two His and two Cys binding to a zinc ion. Variants with three or four Cys that lack the beta strand of the classic motif. Zinc fingers are multidomain with 1-60 tandem zinc finger DNA binding domains and several other domains which may be responsible for dimerization, ligand or protein binding.

Leucine zippers: A motif built of two alpha helices which are kept together by hydrophobic interactions. Each seventh residue is Leu. Dimer formation can be promoted by additional charge interactions. Leu zipper DNA binding proteins are homo- or heterodimers. The C-terminal part contains the dimerization region, whereas the N-terminal part binds to DNA and contains many basic residues.

Helix-loop-helix domains: Similar to Leu zippers, except that a four bundle helix motif holds together basic DNA-binding helices. Dimer interaction.

2. What is histone tail acetylation/deacetylation? Why does it influence binding of histones to DNA? Draw schemes of repressor-directed histone deacetylation and activator-directed histone hyperacetylation!

Histone tail acetylation/deacetylation belongs to chromatin mediated transcription control. With the aid of HDAC, histone deacetylase, the N-terminal sequence of histone H3 becomes unacetylated, which is a form of repressor-directed histone deacetylation. There is also the activator-directed histone hyperacetylation. The activity of a chromatin complex may be repressed by exposing the histone tails to deacetylases. When chromatin gets decondensed the histone tails are exposed and histone acetylases are recruited. Then an activator can bind to the promoter proximal elements which leads the way to the start of transcription.

3. Draw a general scheme of transcription activation (from condensed chromatin to polymerase binding) !

4. What are epigenetic transcription control mechanisms? How does methylation of CpG islands work? How can the DNA methylation pattern be inherited to daughter cells?

Epigenetic transcription control mechanisms are methylation of DNA and histones. DNA methylation of CpG islands is made by DNA methyltransferase. It can block transcription by either direct blocking of TFIID binding or by recruitment of histone deacetylases.

Since DNA methylation is at least sometimes linked to histone modification, conservation of histone pattern in daughter cells might be just a consequence of DNA methylation inheritance. During replication, parental histones are randomly distributed to both daughter chromatides. Modified parental histones might serve as nests for modification of non-parental histones.

If a double helix that is methylated is replicated there will be at least one copy that keeps the methylation and transfers it to daughters.

5. How can transcription factor activity be regulated? In what ways can phosphorylation affect transcription factor activity?

Transcription factor activity can be regulated by covalent modification, e.g. phosphorylation, acetylation, ubiquitination, or by binding to ligands or nuclear receptors. Phosphorylation of transcription factors is due to addition or removal of one or several phosphate groups on Ser, Thr or Tyr residues by a protein kinase or protein phosphatase. It creates stability and can perform localisation and protein-protein interactions. It is involved in DNA binding and transcriptional activity and activation-induced inactivation.

6. What is p53 protein and how does it work? Describe, how differnt mutations in p53 can effect its activity!

P53 is one of the most frequently cited biomolecules. It acts as a tumor supressor which prevents cell division under DNA damaging conditions such as exposure to UV light, etc. Mice lacking p53 have normal development but show predesposition to develop multiple tumors. Half of all people that are diagnosed for various forms of cancer have mutations in the p53 gene.

Under normal conditions p53 is unstable, unphosphorylated and p53 controlled genes are inactive. Under damaging conditions p53 becomes phosphorylated, it is stable and quickly accumulates. When it is transcribes it causes cell death unless DNA is repaired or there is an arrest in the G1 phase.

Mutations in p53 residues that bind to DNA decreases its affinity and thus its effectivity. Residues responsible for a certain loop will change the conformation to an unfunctional loop.

7. What are nuclear receptors and how do they work? What is the difference between homo- and heterodimeric nuclear receptors in terms of binding mode to DNA and mode of action?

Nuclear receptors are transcription factors that get activated by lipid-soluble hormones, i.e. small hydrophobic molecules capable of diffusing freely through plasma and nuclear membranes.

Homodimeric nuclear receptors are made of two identical subunits and bind to inverted DNA repeats because of their two-fold symmetry. Heterodimeric nuclear receptors have direct repeat binding sites. They are made of two different subunits, one of them is always a universal monomer called RXR. Binding specificity is achieved by variable length of spacer nucleotide sequence.

Homodimeric action: In the absence of hormone the nuclear receptor is located in the cytoplasm. When binding to the hormone the receptor gets transported to the nucleus where it bind to the response element.

Heterodimeric action: In the absence of hormone the nuclear receptor binds to the DNA response element and recruits histone deacetylases, transcription is blocked. When the hormone diffuses into the nucleus and binds to the receptor the histone deacetylas gets released and histone acetylase binds instead, transcription is activated.

Non-coding RNAs

1. What is tmRNA? How does trans-translation work?

tmRNA is a housekeeping RNA. It’s a hybrid molecule: half tRNA, half mRNA. tmRNA helps to rescue ribosomes, bound to mRNA which lacks the termination codon. In addition, mRNA adds a degradation signal to nascent protein.

Trans-translation: during translation if the termination codon isn’t reached the ribosome gets stacked upon the reaching of the 3’ end of the mRNA and has to be rescued. The protein made is probably wrong and needs to be degraded. tmRNA is responsible for the rescue. The tRNA part adds an Ala to the growing polypeptide chain while the mRNA enters the ribosome and the synthesis of the polypeptide is continued with the aid of normal tRNA until the termination codon is reached. The ribosome is released and the new protein is degraded due to a signal sequence in the C-terminus.

2. What is dosage compensation? What differences in dosage compensation exist between mammals, C. elegans and Drosophila ? How does roX ncRNA work in Drosophila? How does Xist ncRNA work in mammals?

In animals males and females have different numbers of X chromosomes. To equalize the expression levels from the X chromosome in males and females there is dosage compensation which represses one chromosome. In mammals it deactivates one X chromosome in females. In C. Elegans both X chromosomes in females are repressed, to an equal amount of the single X chromosome in males. In Drosophila the X chromosome in males in hyperactivated to equal the two chromosomes in females.

roX ncRNAs are expressed only in males which activates the MSL (male specific lethal) complex which acetylates H4 histones on the X chromosome thereby increasing the transcription level.

Xist ncRNAs silence X chromosomes by recruiting a specific histone isoform which maintains the chromosome in its inactive state. Xist also recruits deacetylases and methylases. The chromatin becomes condensed by the histone deacetylases. Xist is regulated by Tsix, its anti-sense sequence, which can basepair to it and thereby regulate it.

3. How does anti-sense mechanism of translational regulation work? (Do not tell about RNA interference here!) Can you give examples of both translation activation and inactivation by anti-sense mechanisms?

Anti-sense mechanism is when the ncRNA binds to the target mRNA. Translation of human HFE genes are downregulated by anti-sense RNA. DsrA RNA in E.coli activates ribosome binding to stress-response sigma factor rpoS mRNA. The ribosome binding site is blocked by base-pairing. When DsrA RNA binds the ribosome binding site is accessible.

4. What are ribozymes? What are the known catalyzed reactions of naturally occuring ribozymes? Can synthetic ribozymes catalyze some other reactions?

Ribozymes are ribonucleic acid enzymes, or RNA molecules with catalytic properties. It can be for example cleaving or hammerhead. Naturally occuring ribozymes are found mainly within self-splicing intrones and in RNA encoded parasites (satellites and viroids). They are limited to cleavage and ligation of RNA. Their catalytic activity is much lower than of analogous protein enzymes. Synthetic ribozymes can catalyse other reactions than just cleaving and ligation.

5. What is RNA interference? How does it work? What are the differences of RNAi in fungi, plants, worms and Drosophila and mammals?

RNA interference is a natural biological mechanism for silencing genes. It is a revolutionary new technology to knock down gene expression in eukaryotic cells. The silencing takes place in the cytoplasm (?) and occurs post-transcriptionally.

The injection of dsRNA into C.elegans resulted in the silencing of a gene complementary to the dsRNA. It leads to mRNA cleavage, all mRNA will disappear.

It is a highly evolutionarily conserved property in eukaryotes. It is a defense against dsRNA-containing viruses. It may stabilise the genome and control cellular development.

RNAi in fungi, plants and worms has a systematic nature of silencing, is inheritable. They can replicate silencing RNA with RNA-dependent RNA pol. In Drosophila and mammals the RNAi is non-inheritable and they have cell-autonomous silencing. There is no indication of silencing RNA replication.

6. Tell me about RNA world! Where does the RNA polymerase, made of RNA come from? What are its drawbacks, compared to polymerases, made of protein?

In the RNA world there was no DNA that transfered information and carried information. RNA was the only means of storing and transfering information. There must have been an RNA molecule which was capable of making RNA itself, or in other words an RNA polymerase made of RNA. So far, such a polymerase is not known to exist in nature, but a synthetic one has been made. Its extension time is ~14 nucleotids in 24 hours. After the RNA world the result was ribozymes able to catalyse peptide bond formation and other chemical reactions. Then proteins began to take over the enzymatic activities. However, before RNA there was what? The synthetic polymerase is 165 nt long which is far too long to emerge accidentally.

RNA is more unstable than DNA.

Viruses

1. What are viruses? What are the theories of virus origins?

Viruses are submicroscopic intracellular parasites. Virus particles are made from assemblies of already made components and don’t grow or divide. They lack the genetic information for generation of metabolic activity and protein synthesis, i.e. they can’t survive on their own.

There are three theories on where they came from: Regressive theory suggests that viruses are a degenerate form of intracellular parasites. Mitochondria and chloroplasts are suggested to have been derived from intracellular parasites. However they don’t have their worn rRNAs or synthesis. Progressive theory suggests that they are cellular RNA and DNA components, i.e. normal nucleic acids that have gained the ability toreplicate autonomously and evolve. They came from plasmids or transposable elements. Then they evolved coat proteins and transmissibility. Coevolution theory suggests that viruses coevolved with life and their evolution might go all the way back to the RNA world.

2. How is the virus particle organized? What is the difference between enveloped and non-enveloped viruses? What is nucleocapsid? What are the two types of nucleocapsids?

The virus particle encloses genomic nucleic acid and is a polymer derived from one or a few different kinds of monomers. The protein transcribed is not long enough to enclose the nucleic acid  polymer.

Enveloped viruses have their nucleocapsid (that contains the genome) encapsulated within a lipid bilayer with enveloped proteins. Non-enveloped viruses only have the inner nucleocapsid (that contains the genome).

The nucleocapsid is the viral nucleic acid enclosed in the protein shell. They can be either filamentous, (e.g. tobacco mosaic virus) which is a helical structure with a slight axial rise each turn, or icosahedral, made up by 60 equal subunits inside an 18-sided diceshaped cube.

3. How are viruses classified according to their genomes?

Viruses are classified according to their genomes and ways of replication. There are doublestranded DNA viruses, Singlestranded (+) sense DNA, Doublestranded RNA, Singlestranded (+) sense RNA, Singlestranded (-) sense RNA, Singlestranded (+) sense RNA with DNA intermediate in the lifecycle, Partial doublestranded gapped DNA with RNA intermediate.

4. Describe the virus life cycle in general!

Initiation phase: the virus attaches to the host cell, penetrates the cell wall and finally uncoates. Bacteriophages avoid penetration and uncoating by injecting their nucleic acids straight into the cell.

Replication phase: consists of nucleic acid replication, mRNA synthesis, protein expression and assembly.

Release phase: the virus exits the cell and matures, e.g. rearranges its nucleocapsid, etc. In enveloped viruses the assembly can be associated with release.

5. Describe the life cycle of HIV!

HIV has two exactly identical copies of the genome. It penetrates the cell wall of the host cell and injects its reverse transcriptase. It transcribes its viral RNA into the hosts DNA. Retroviruses are ejected from the nucleus. During maturation gag and gag-pol polyproteins are cleaved into active units. This rearranges the virion/nucleocapsid structure and makes the particle infectous.

6. What are satellite viruses? What types of sattelite viruses you know?

Satellites are virusoids, small RNA molecules, absolutely dependent on the presence of another virus. They often cause different symptoms than their host virus. Most known satellites are connected to plants, but some are dependent on animal viruses.

There are four types: A, an RNA molecule with more than 700 nts that encodes its own capsid protein. B, an RNA molecule with more than 700 nts that encodes a non-structural protein. C, a linear RNA molecule of less than 700 nts that doesn’t encode any proteins. D, a circular RNA of less than 700 nts that doesn’t encode any proteins. There are also several DNA satellites.

7. What are viroids? How is viroid RNA replicated? What is hepatitis delta virus?

Viroids are very small rodlike RNA molecules with a high degree of secondary structure. They don’t encode any proteins and unlike satellites they are not dependent on the presence of another virus.

Viroids utilise cellular RNA polymerases for their replication. It is performed by rolling circle mechanism. The pol II rolls around the entire circle (like a plasmid). The resulting long RNA is cut into pieces and ligated. In a sense some viroids are ribozymes.

Hepatit delta virus is a chimeric molecule, half viroid, half satellite. It has the rodlike RNA molecule and the rolling circle replication and selfcleaving activity of the viroids and it encodes a protein and is dependent on another virus (HBV) and has a genome with 1640 nts like the satellites.

8. What are prions? What kinds of prions do you know? What is PrPc and PrPSc? How does PrPc gets converted to PrPSc?

Prions are chronic, progressive and fatal infections of the nervous system. The infectious agent is protein only, without the presence of any nucleic acid. The main known infections are scrapie, mad cow disease and Creutzfeldt-Jakob disease.

PrPc is cellular, the normal variant of the protein of unknown function expressed in nervous tissue. It is fatal. Lacks immunodefence system.

PrPsc (scrapie) is the same protein which has undergone severe structural rearrangements forming unsoluble beta sheet rich fibrils similar to those caused by Alzheimer’s disease. PrPsc can catalytically convert PrPc to PrPsc. PrPsc is extremely stable, it can survive temperatures over 100 degrees.

Talal's lectures about signal transduction

(a few more questions will come)

1. What are the basic components of the GPCR signaling mechanism?

The crucial function of G-protein coupled receptors are vision and smell. The receptor is a membrane protein with seven transmembrane helices. When the receptor is activated it binds and activates various related trimeric proteins called G-proteins. The active G-protein binds to a signal molecule which allows it to bind to an enzyme which activates it.

Upon ligand binding the receptor is activated. The G-protein is converted from GDP to the active form GTP. The second messengers, cAMP etc, get synthesised. The transcription factors get activated. Ligands can be e.g. adrenalin, prostaglandine and rhodopsin.

2.Where can regulation occur in a signaling pathway?

Integration of various pathways is important in order to get the appropriate respinse. Activation of a pathway might change the cell’s ability to react to other signals.

Several receptors use the same pathway components, yet the outcome is different. Reasons are that the strength or duration of signal governs the outcome. Downstream elements are different depending on cell type. Convergence of more than one activated pathway determines the final response.

Signal pathways regulation results in degradation of the second messenger, deactivation of the signal transduction protein and desensitisation of the receptors. There are also inducible antagonists (drugs that bind to the receptor in order to block the binding of the natural hormone) that can act either extra- or intracellularly.

3. In which way do RTKs activation differs from the GPCR signaling activation ?

Receptor Tyrosine Kinases are transmembrane proteins that have a tyrosine kinase activity on the cytosolic side. Ligands are growth factors and insulin and so on.

After the first phosphorylation the kinase activity is increased due to conformational changes in the catalytic site. Increased activity leads to phosphorylation of other specific tyrosine residues. The phosphorylated tyrosine residues serve as anchors for secondary proteins needed for signal transduction. They are targets for SH2 and PTP domains.

Adapters are usually needed to activate the pathway. Activation is done through activation of a small GTPase protein called Ras.

Activation of the receptor leads to phosphorylation of Tyr residues. Adapter molecules anchor to the phosphorylated Tyr residues. Some need a second adapter to bind GRB2. The complex of GRB2 and SOS and a GDP bind to the phosphorylated Tyr once the receptor is activated. All this activates the Ras, an GTPase which starts the signaling.

Conclusion: RTKs need to be dimers to activate a pathway. There is phosphorylation of the cytosolic domain when the ligand is bound. RTKs are linked to Ras via an adapter, GRB2, and SOS. When active Ras, signalling occurs. Different from GPCRs which aren’t dimers and less complicated in my opinion.

4. What is the use of Scaffolds?

Scaffold proteins aid signaling. They bind multiple signaling proteins together in a complex. They hold the complex at a particular location. They organise groups of interacting proteins in order to enhance speed, precision and efficiency of the transduction. They have been documented in yeast, C.elegans and Drosophila but never in mammalian cells.

Organisation of signaling proteins into signaling complexes eliminates the risk of cross-talk between pathways that use the same signaling proteins. When the ligand dissociates, the complex is disassembled.

5. In the case of ion channels, how does the signal starts and propagates?

Ion channel receptors are the third largest class of receptors. The ligands include acetylcholine and glutamate. They act on K+, Na+ and Ca++ transport.

In the motor neuron: Action potential. Voltage gated Ca++ influx leads to release of acetylcholine. In the muscle cell membrane: A local depolarisation of the membrane leads to influx of Na+ through voltage gated Na+ channels. Inside the muscle cell: T-tubules are reached by depolarisation, which is also sensed by the voltage gated Ca++ channels. There is a release of Ca++ from the Sarcoplasmic reticulum which leads to a higher concentration of Ca++ in the cytosol. The muscle contracts.

Jan's lectures about prokaryotic genomes

1. Livet brukar delas in prokaryoter och eukaryoter. Nämn tre egenskaper som skiljer dessa celltyper åt.

Prokaryoter saknar kärna med membran, organeller med membran, fagocytos (förmåga att svälja andra partiklar), sex (meios och mitos), linjära kromosomer med histoner och introner.

2. Redogör för vilka huvudtyper av genetisk information genomet hos en typisk prokaryot innehåller.

De innehåller typiskt 90% proteinkodande gener, öppna läsramar som antas vara kodande. De har även gener som kodar för stabila RNA (<2%) och 5-20% icke-kodande områden såsom promotorer, reglersekvenser och pseudogener.

3. Vad är GC-skew och hur detekteras det? Vad orsakas det av och vad kan man använda det till? Motivera dina svar.

GC-skew är mönster i genomet. Halten G och C varierar mellan de två strängarna. GC-skew definieras som (C-G)/(C+G). Man kan skapa en graf som plottar GC-skew mot antalet baser för att kunna se var det finns överskott respektive underskott av C. Det orsakas av olika mekanismer för replikationen av leading och lagging strand som medför olika mutationsmönster. Det används för att identifiera startpunkten för replikationen.

4. Vad kännetecknar typiska kromosomer och plasmider? Vilka avvikelser förekommer? Motivera dina svar.

Kromosomer och plasmider innehåller det genetiska materialet hos prokaryoter. Plasmider är ofta mycket mindre än kromosomer. Det finns ofta endast en kromosom medan plasmiderna kan vara många i antal. De är ofta cirkulära. Det finns undantag hos prokaryoter som har flera stora DNA molekyler som man skulle kunna kalla kromosomer. Även linjära bakteriella kromosomer har hittats. Kromosomer och plasmider är nära kopplade till varandra, men man vet inte om plasmiderna själva kan stå för smitta. T ex återfinns borrelias plasmider i alla kliniska isolat som undersökts från smittade personer. Det gör att plasmiderna gränsar mot att kallas kromosomer pga deras nödvändiga funktion för bakterien.

Kolera är också ett undantag som har två cirkulära kromosomer, en större och en mindre.

5. Redogör för en bakterie där arvsmassan återfinns i en ovanlig konfiguration. Namnge bakterien och nämn hur den lever, samt hur arvsmassan är organiserad och vad som skiljer detta mot den typiska bakterien.

Vibrio cholera skiljer sig från andra bakterier därför att den har två cirkulära kromosomer istället för endast en. De essentiella generna är överrepresenterade på den större kromosomen men finns även på den mindre. Gener involverade i metabolittransport och metabolism av socker och energi är överrepresenterade på den mindre kromosomen. De flesta patogena generna finns på den större kromosomen som har normal replikering. Den mindre kromosomens replikering är okänd.

Kolera orsakas av denna bakterie som växer på tunntarmen och producerar koleratoxin som stimulerar vattenutsöndring vilket leder till kraftig diarré för människor.

6. Vad är operoner och vad kännetecknar dem? Spekulera varför denna geniala uppfinning inte är konserverad mellan arter!

Operoner är unika för prokaryoter. De är en följd av gener på genomet som uttrycks tillsammans, dvs ett långt mRNA som innehåller information till flera proteiner som syntetiseras från DNA. I E.coli har sådana gener ofta relaterad funtion, men detta stämmer ej för andra arter. Det är praktiskt för reglering av genuttryck. Endast ett fåtal operoner, tex de som kodar för ribosomala proteiner, är konserverade mellan avlägsna släktingar. Många operoner innehåller även gener utan funktion.

7. Beskriv hur genordningen i prokaryota genom kan studeras. Vilka mönster brukar uppkomma och hur kan detta förklaras?

Man plottar vilka gener som finns gemensamt i två genom och på vilken position de förekommer. Man anger med punkt om de har samma riktning och cirkel om motsatt. Förändringarna brukar ofta vara centrerade kring oriC, dvs starten för replikationen.

En uppåtgående kurva tyder på två morfologa gener. En korsliknande kurva tyder på samma gener men i olika ordning. Många förändringar sker under replikationen. Rekombinationer av avlägsna delar av kromosomen är en möjlighet. Organisationen hos prokaryota genom ändras snabbt över evolutionär tid.

8. Hur stor är variationen av den totala GC-halten mellan olika prokaryota genom? Hur korrelerar den totala GC-halten i genomen med GC-halten i de olika delarna av genomet (protein-kodande gener, rRNA, tRNA, respektive spacer områden)? Rita och beskriv. Hur kan detta förklaras i evolutionära termer?

Halten av baserna varierar mellan olika genom. Eftersom basparning sker är halterna av G=C och A=T. Sammansättningen av nukleotiderna i ett genom anges som en GC-halt. Den varierar från 25-75%. Eftersom baspar med GC har tre vätebindningar är de mer stabila än AT med endast två.

Olika delar av genomet motsvarar olika mängd av GC-halt. Variationen är störst i icke-kodande regioner och minst i RNA-kodande regioner. Regioner under stark selektion påverkas minst av GC-halten i genomet i stort. Hos Rickettsia som har ~30% GC-halt sker de flesta mutationer till A och T. Mutationer i proteinkodande gener selekteras bort om de leder till ändringar i funktion. Vad man kan säga är i vart fall att den tredje positionens mutationer påverkas minst och den andra positionen mest.

9. Synonyma kodon är olika kodon som kodar för samma aminosyra. Det har visat sig att synonyma kodon inte används slumpmässigt i många arter. Vad kan detta bero på? Motivera ditt svar.

Synonyma kodon skiljer sig oftast endast i tredje position. GC-halten är ofta hög i synonyma tredje positioner. Högt uttryckta gener använder färre kodon än det förväntas enligt GC-halten. Det orsakas antagligen av selektion för effektiv translation, vissa kodon är bättre.

10. Hur stora (antal bp) är prokaryota genom? Finns det något samband mellan livsstil och genomstorlek hos prokaryoter? Motivera ditt svar.

Storleken på prokaryota genom varierar från 0.5 till 10 Mbp motsvarande 500 till 10000 gener. De mindre genomen tillhör prokaryoter som lever som symbionter eller parasiter, dvs de kan inte överleva utan en värdcell. Det är ett linjärt förhållande mellan genomstorlek och antal gener. Större genom innehåller alltså fler livsuppehållande gener vilket medför att sådana prokaryoter har förutsättningar att klara sig på egen hand. Tex har Rickettsia, som är en obligat parasit, mycket få gener som kodar för metaboliska proteiner medan E.coli som är mer anpassningsbar och ändrar sig efter de varande förhållandena måste utnyttja de metaboliter som finns tillgängliga.

11. Rickettsia har förlorat en stor del av den genetiska informationen som fanns hos dess anfader. Hur har denna förminskning av genomet påverkat olika kategorier av gener? Hur korrelerar detta med livsstilen hos Rickettsia? Motivera ditt svar.

Rickettsia är en obligat intracellulär parasit som orsakar tyfus. Förhållandet mellan antalet gener i olika funtionella grupper avspeglar detta levnaddsätt. Det har få gener som kodar för proteiner med metabolisk funtion eftersom det beror av en värdcell. Rickettsia har flera pseudogener och en låg andel kodande DNA, 24% av genomet är icke-kodande och det finns många regioner utan funktion. Det finns en hypotes att pseudogenerna motsvarar gener som inte längre används pga ändade livsstil och de långa icke-kodande regionerna är tidigare inaktiverade gener som inte längre var identifierbara.

12. Vad är horisontell genöverföring och hur kan den gå till hos prokaryoter?

Horisontell genöverföring är överföring av genetiskt material mellan avlägsna släktingar. Genom konjugation byter bakterier information genom fysisk kontakt, så pass att hela genomet kan överföras i en följd. Genom transduktion överförs små fragment via bakteriofager. Genom transformation tar prokaryota celler upp DNA-molekyler (plasmider) från omgivningen troligen i födosyfte. Bakterier lever i heterogena miljöer omgivna av mycket genetiskt material vilket gör att det är lätt för dem att ta upp gener.

13. Horisontell genöverföring kan detekteras genom att identifiera gener med avvikande sekvensmönster (GC-halt) inom ett genom. Dessa gener antas ha tagits upp från en annan art. Varför kan denna metod enbart identifiera gener som upptagits relativt nyligen evolutionärt sett? Motivera ditt svar.

Eftersom horisontell genöverföring baseras på att GC-halten hos nya tillskott till ett genom skiljer sig mycket från det övriga genomet kan det bara identifiera nyupptagna gener därför att GC-halten hela tiden genomgår mutationer. Det är oklart hur man ska definiera avvikelse. Nu bedöms bara första och tredje position som är de positioner som har högst GC-halt.

14. BLAST-sökningar har ofta används för att identifiera horisontella genöverföringar. Vad är det största problemet med denna metod? Motivera ditt svar.

Oväntad bästa sekvenslikhet har förklarats med horisontell genöverföring, t ex att en människogen får bästa träff i en bakteriegen sägs vara pga en genöverföring från människa till bakterie. I en BLAST-sökning antar man helt enkelt att den bästa träffen beror på en genöverföring om den bästa sekvensen inte är den förväntat närmaste släktingen. MEN bästa sekvenslikhet innebär inte närmast släkt.

15. Hur identifieras en horisontell genövering i ett fylogenetiskt träd baserat på en enskild sekvens?

Med fylogenetiska metoder fås relationerna mellan många sekvenser samtidigt. Mottagaren av en genöverföring återfinns inom gruppen som var givare i överföringen, i en ideal situation. Fylogenetiska metoder är skapade för att finna evolutionära släktskap mellan sekvenser och är därför överlägsna BLAST för att visa och motbevisa genöverföringar. Tyvärr är fylogenetiska metoder inte heller perfekta. Det finns risk att dra felaktiga slutsatser från resultat som baseras på systematiska fel i sekvenserna, t ex GC-halten i genomet. Ofta kan flera biologiska förlopp vara tänkbara förklaringar till ett träd. Biologisk expertis krävs för att tolka informationen.

16. Genom att jämföra förekomsten av gener i närbesläktade stammar är det relativt enkelt att identifiera gener som nyligen tagits upp eller förlorats. Redogör för ett släkte av bakterier där sådana studier har gjorts och vad de i stora drag visade.

En sådan studie gjordes på Prochlorococcus, den minsta kända fotosyntetiska syrebildande organismen, även känd som cyanobakterien. Två stammar sekvenserades. Av 1716 respektive 2275 gener återkommer 1350 gener i båda genomen. Med hjälp av genordningskartläggning ser man att de unika generna är fördelade över hela genomet. Omorganisationerna är symmetriska runt oriC, startpunkten för replikation. Stammarna är anpassade för olika miljöer, t ex har de två olika djupoptimum i havet. Skillnaderna i genomet återspeglar antagligen de olika förutsättningarna i de olika miljöerna.

17. Horisontell genöverföring är en viktig process under evolutionen av prokaryoter. Hur påverkas olika typer av gener och hur kan eventuella skillnader förklaras?

Gener som kodar för metaboliska proteiner med få interaktioner genomgår ofta horisontell överföring. Gener som kodar för proteiner med många interaktioner med andra proteiner i cellen, t ex translations- och transkriptionsassocierade proteiner, genomgår genöverföring mer sällan. Proteiner med få interaktioner är lätt utbytbara medan proteiner med många interaktioner har evolverat tillsammans med proteinerna de interagerar med och är därför svårare att byta ut. Arter som lever tillsammans överför gener i större utsträckning. Intracellulära parasiter och symbionter som sällan kommer i kontakt med andra prokaryoter påverkas lite av genöverföringar. Gener som sällan byts ut är lämpliga att använda som markörer i fylogenetisk analys.

Microtubules

1. In vitro tubulin polymerization assays show that after a period of rapid polymerization the amount of polymerized mictotubules finally reaches a constant level. a) Does it mean that these microtubules are stable and do not gain or lose subunits? Describe this steady state. b) Same kind of steady state occurs in spindle microtubules in metaphase. How can this be visualized experimentally?

At the critical concentration, Cc, the rate constant for addition of monomers will equal the rate for loss of monomers. The number of monomers that add to the polymer per second will be proportional to the concentration of the free subunit. The subunits will leave the polymer end at a constant rate that doesn’t depend on the concentration. As the polymer grows, subunits are used up, and the concentration is observed to drop until it reaches a steady value, the critical concentration. If both ends of the polymer are exposed the critical concentration at both ends is different due to differences in composition and GTP hydrolysis activity. If the plus end has a value above the Cc and the minus end is below the Cc there will be a steady state since the minus end is more stable and grows slower than the plus end. The polymer maintains a constant length but is growing from one end simultaneously as it degrades from the other. Microtubules form the mitotic spindle. In the metaphase the two sister chromatides are separated. The microtubuli length remains the same during metaphase, but there is a constant poleward flux of subunits. By labelling the MTs with a substochiometric amount of fluorescent subunits so that the MT appears as speckled the movement of fluorescent dots can be followed in a timelaps series. They will appear constant.

2. A key property for the function of microtubules in the cell is their ability to display dynamic instability. (a) What is dynamic instability? (b) Explain the molecular basis for dynamic instability.

Dynamic instability is the fact that the MTs grow faster from the plus end and slower from the minus end. The plus end can switch between phases of slow growth and rapid shrinkage. In vivo shrinkage can lead to either rescue by growing of the other end or catastrophe by disappearing. The GTP hydrolysis cycle is connected to movement. When the MT grows it has a cap of GTP at the end. Hydrolysis of GTP leads to release of energy that can be used for movement. MT associated proteins such as MAP and Katanin may be stabilisers or destabilisers. Destabilisers increase the catastrophe rate and induce depolymerisation. Stabilisers promote rescue.

3. (a) What cellular structures organize microtubules in an interphase cell? (b) What are the typical components of these structures in an animal cell? (c) What is the molecular basis of their ability to organize cellular microtubules?

The MTOC, MT organising centre, is from where the MT grow, aka the centrosome. It contains a pair of microtubuli based structures called centrioles, without known function. The centrosome is a collection of MT-associated proteins. There is also the pericentriolar matrix which contains proteins that organise MTs.

4. Kinesins of different types move along microtubules in different directions and carry various cargo. (a) Which part of the kinesin determines which cargo it will carry? (b) Which part of the kinesin determines the direction of movement? Describe an experiment how to verify that the --- part of different kinesins is responsible for determining the direction. (c) Give an example of kinesin-powered transport in a cell.

The sequence of the unique tail of the kinesin determines which cargo it will carry. It can have both a plus and a minus directed motor. It can swap tail and thereby change cargo. They are divided into cytosolic and mitotic kinesins, depending on the type of cargo they carry. Kinesin-powered transport can be membrane enclosed organelles such as ER and Golgi complexes, secretory vessicles, endosomes, lysosomes, mitochondria and pigment granules.

5. Major rearrangements in the microtubule network occur during cell cycle progression. (a) What is the main difference between interphase and mitotic microtubules? (b) Which proteins influence microtubule dynamics in the cell and how? (c) How can the activity of these proteins be regulated in a cell-cyle-dependent manner?

Interphase is a stage in mitosis. It is a very strangely posed question. Catastrophy rates are increased by Op18 and KinI kinesins. These rates are counteracted by plus end stabilizing MAPs. CLIP170 and EB1 stabilise and reduce catastrophes. XMAP215 binds to the plus end and increases the growth rate but doesn’t reduce catastrophes. MAP activity can be suppressed by phosphorylation. XMAP215 is regulated by cyclin-dependent kinases.

6. In anaphase the sister chromatids are separated and segregated into opposite cell parts. (a) What happens with chromosomes and the mitotic spindle in Anaphase A and Anaphase B, respectively? (b) What is the role of dynamic microtubules in these movements? (c) Describe the molecular mechanisms behind this ability of microtubules to act as a molecular motor and move chromosomes in anaphase?

In anaphase the glue that holds the sister chromatids together is dissolved and they move apart. In anaphase A the chromosomes separate and move towards opposite spindle poles due to the shortening of kinetochore MTs. In anaphase B the two spinde poles move apart bringing the chromosomes into new daughter cells. Depolymerisation occurs at the minus ends. The kinetochores chew up the MT plus ends as they move towards the pole. Plus end directed cross-linking motors decrease the overlap of antiparallel microtubules and contribute to spindle pole separation. The polar MTs lengthen at their plus ends. MTs can move things without motor proteins. The force generated by polymerization/depolymerization of the MTs is enough to move chromosomes during mitosis or to position the spindle in the cell.

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