Molecular Biology Wk 9 Flashcards
Proteins Enter the Nucleus Through Nuclear Pores
The signal sequence that directs a protein from the cytosol into the nucleus, called a nuclear localization signal, typically consists of one or two short sequences containing several positively charged lysines or arginines. The nuclear localization signal on proteins destined for the nucleus is recognized by cytosolic proteins called nuclear import receptors.
A nuclear pore is a large, elaborate structure composed of a complex of about 30 different
proteins .
Many of the proteins that line the nuclear pore contain extensive, unstructured regions in which the polypeptide chains are largely disordered.
Nucleic Acids
Nucleic acids are macromolecules constructed out of long chains ( strands ) of monomers called nucleotides . There are two types of nucleic acids found in living organisms, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids function primarily in the storage and transmission of genetic information, but they may also have structural or
catalytic roles.
Each nucleotide in a strand of RNA consists of three parts (FIGURE ):
(1) a five‐carbon sugar, ribose;
(2) a nitrogenous base (so called because nitrogen atoms form part of the rings of the
molecule);
(3) a phosphate group.
The sugar and nitrogenous base together form a nucleoside , so that the nucleotides of an RNA
strand are also known as ribonucleoside monophosphates.
Nucleotides of RNA.
The nucleotides of RNA contain the sugar ribose, which has a hydroxyl group bonded to the second carbon atom. In contrast, the nucleotides of DNA contain the sugar deoxyribose, which has a hydrogen atom rather than a hydroxyl group attached to the second carbon atom.
Nucleic Acids
A strand of RNA (or DNA) contains four different types of nucleotides
distinguished by their nitrogenous base:
Pyrimidines
Purines
Pyrimidines are smaller molecules, consisting of a single ring;
purines are larger, consisting of two rings
Nucleic Acids
RNAs contain two different purines - adenine and guanine , and two different pyrimidines - cytosine and uracil . In DNA, uracil is replaced by thymine , a pyrimidine with an extra methyl group attached to the ring (Figure). The two strands of DNA in the double helix structure are complementary; A binds with T and G binds with C to form units called base pairs.
Alternative Forms of DNA
At the time when Watson and Crick performed their analysis, two forms—A-DNA and B-DNA— were known. In comparison to B-DNA, A-DNA is slightly more compact.While it is a right-handed helix, the orientation of the bases is somewhat different—they are tilted and displaced laterally in relation to the axis of the helix. It seems doubtful that A-DNA occurs in vivo (under physiological conditions).
Still other forms of DNA right-handed helices have been discovered when investigated under various laboratory conditions. These have been designated C-, D-, E-, and most recently P-DNA. C-DNA is found under even greater dehydration conditions than those observed during the isolation of A- and B-DNA. Two other forms, D-DNA and E-DNA, occur in helices lacking guanine in their base composition. And most recently, it has been observed that if DNA is artificially stretched, still another conformation is assumed, called P-DNA (named for Linus Pauling). Another form of DNA, called Z-DNA, was discovered in 1979, when a small synthetic DNA oligonucleotide containing only G-C base pairs was studied. Z-DNA takes on the rather remarkable configuration of a left-handed double helix.
DNA Supercoiling
In 1963, Jerome Vinograd and his colleagues at the California Institute of Technology discovered that two closed, circular DNA molecules of identical molecular mass could exhibit very different rates of sedimentation during centrifugation. Further analysis indicated that the DNA molecule sedimenting more rapidly had a more compact shape because the molecule was twisted upon itself (FIGURE a, b). DNA in this state is said to be supercoiled. Because supercoiled DNA is more compact than its relaxed counterpart, it occupies a smaller volume and moves more rapidly in response to a centrifugal force or an electric field (Figure c).
Cells rely on enzymes to change the supercoiled state of a DNA duplex. These enzymes are called topoisomerases because they change the topology of the DNA.
RNA types and functions
LOOK AT GOONDOTES
Nucleotides
Nucleotides are not only important as building blocks of nucleic acids, they also have
important functions in their own right.
Adenosine triphosphate (ATP) - most of the energy being put to use at any given moment in any living organism is derived from the ATP.
Guanosine triphosphate (GTP) is another nucleotide of enormous importance in cellular activities. GTP binds to a variety of proteins (called G proteins) and acts as a switch to turn on their activities
Analytical Techniques: Molecular Hybridization
• The denaturation/renaturation of nucleic acids is the basis for one of the most useful techniques in molecular genetics—molecular hybridization. Duplexes can be re-formed between DNA strands, even from different organisms, and between DNA and RNA strands. For example, an RNA molecule will hybridize with the segment of DNA from which it was transcribed. As a result, nucleic acid probes are often used to identify complementary sequences. The technique can even be performed using the DNA present in chromosomal preparations as the “target” for hybrid formation. This process is called in situ molecular hybridization. Single-stranded DNA or RNA is added (a probe), and hybridization is monitored. The nucleic acid that is added may be either radioactive or contain a fluorescent label to allow its detection. In the former case, autoradiography is used.
Process of creating a hybrid strand of DNA/RNA
The two strands of a DNA molecule are denatured by
heating to about 100°C = 212°F (a to b). At this temperature, the complementary base pairs that hold the double helix strands together are disrupted and the helix rapidly dissociates into two single strands.
The DNA denaturation is reversible by keeping the two single stands of DNA for a prolonged period at 65°C = 149°F (b to a). This process is called DNA renaturation or hybridization.
Similar hybridization reactions can occur between any single stranded nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the renaturation process, the RNA competes with the coding DNA strand and forms double-stranded DNA/RNA hybrid molecule (c to d). These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe.
FISH (fluorescent in situ hybridization)
Figure illustrates the use of a fluorescent label. A short fragment of DNA that is complementary to DNA in the chromosomes’ centromere regions has been hybridized. Fluorescence occurs only in the centromere regions and thus identifies each one along its chromosome. Because fluorescence is used, the technique is known by the acronym FISH (fluorescent in situ hybridization). The use of this technique to identify chromosomal locations housing specific genetic information has been a valuable addition to geneticists’ repertoire of experimental techniques.
Applications of Fluorescence In Situ Hybridization Technology in Malignancies
Fluorescence in situ hybridization (FISH) has greatly enhanced the field and enabled a more precise determination of the presence and frequency of genetic abnormalities. The advantages of FISH compared to standard cytogenetic analysis are that FISH can be used to identify genetic changes that are too small to be detected under a microscope, does not require cell culture, and can be applied directly on fresh or paraffin-embedded tissues for rapid evaluation of interphase nuclei.
FISH for bacterial pathogen identification
LOOK AT GOODNOTES
Telomeres Solve Stability and Replication Problems at Eukaryotic Chromosome Ends
The presence of linear DNA “ends” on eukaryotic chromosomes creates two potential problems: 1. The double-stranded “ends” of DNA molecules at the termini of linear chromosomes resemble the double-stranded breaks (DSBs) that can occur when a chromosome becomes broken internally as a result of DNA damage. Such double-stranded DNA ends are recognized by the cell’s DNA repair mechanisms that join the “loose ends” together, leading to chromosome fusions and translocations. If the ends do not fuse, they are vulnerable to degradation by nucleases.
2. The second problem occurs during DNA replication, because DNA polymerases cannot synthesize new DNA at the tips of single-stranded 5 ends.
In 1978, Elizabeth Blackburn and Joe Gall reported the presence of unexpected structures at the ends of chromosomes of the ciliated protozoan Tetrahymena. Later Elizabeth Blackburn and her graduate student, Carol Greider discovered telomarase. For her discovery of telomerase and its action, Elizabeth Blackburn received the Nobel Prize for Medicine and Physiology in 2009.
These repeat regions make up the chromosome’s telomeres. In humans, the telomeric sequence 5’-TTAGGG-3’ is repeated several thousand times and telomeres can vary in length from 5 to 15 kb.
Telomerase
After replication is completed RNA primers are removed. The resulting gaps within the new daughter strands are filled by DNA polymerase and sealed by ligase. These internal gaps have free 3’-OH groups available at the ends of the Okazaki fragments for DNA polymerase to initiate synthesis. The problem arises at the gaps left at the 5 ends of the newly synthesized DNA [gaps (b) and (c) in Figure]. These gaps cannot be filled by DNA polymerase because no free 3’-OH groups are available for the initiation of synthesis. The solution to this so-called end-replication problem is provided by a unique eukaryotic enzyme called telomerase.
Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells
