7. Molecular Genetics + Flashcards
(52 cards)
1
Q
Enzyme that unwinds the DNA during replication?
A
- Helicase unwinds the DNA, forming a Y-shaped replication fork
- single stranded binding proteins attached to each strand of uncoiled DNA to keetp them separate.
2
Q
Topoisomerases
A
- break and rejoin the double helix, allowing prevention of knots.
3
Q
Direction of movement of DNA polymerase?
A
- moves 3’ to 5’, synthesized new strand 5’ -> 3’
4
Q
Leading strand vs. lagging strand
A
- leading strand works continuously as more DNA unzips (synthesized 5’ -> 3’
- lagging strand - DNA polymerase has to go back to replication fork and work away from it. produces fragments called okazaki fragments
5
Q
DNA ligase
A
- connect okazaki fragments
6
Q
Primase
A
- enzyme that creates a small strip of RNA primer off of which dna polymerase can work since it can only add to existing strand
- DNA replication requires an RNA primer
- every okazaki fragment has an RNA primer. These RNA strips are later replaced with DNA by DNA polymerase I
7
Q
DNA polymerases
A
- dna polymerase I replaces BPs from primer and does DNA repair
- DNA polymease 3 is pure replication. polymerse 3 can do some proofreading
- in all cases of repair, ligase must come in to seal the backbone afterward
8
Q
Energy for elongation
A
Provided by two additional phosphates that are attached to each new nucleotide. Breaking the bonds holding the two extra phosphates provides the chemical energy for the process.
9
Q
DNA and RNA are polymers of nucleotides, which consist of…
A
nitrogen base, sugar, and a phosphate
10
Q
RNA structure:
A
- mRNA; linear
- tRNA: “clover-leaf” shaped
- rRNA: globular
11
Q
Semiconservative replication
A
- Replication of DNA results in a double stranded molecule containing one “new” strand and an old strand (template) from the original DNA molecule.
12
Q
Direction of movement of DNA polymerase:
A
3’ —> 5’ in each template strand
- complement strand grows in antiparallel, 5’ –> 3’ direction
13
Q
Leading strand
A
- replication occur continuously 3’ –> 5’ as DNA polymerase follows the replication fork
14
Q
Lagging strand
A
- 5’ –> 3’ template strand
- DNA polymerase has to work in direction away from replication fork, creating okazaki segments that are put together by DNA ligase. more time to assemble –> lagging strand
15
Q
Primase
A
- DNA polymerase is able to attach nucleotides only to already existing complementary strand. Therefore, to initiate a new complementary strand, another enzyme, primase, begins replication with a short segment of RNA (not DNA) nucleotides called RNA primer
- the leading strand and every okazaki segment must begin with an RNA primer.
The RNA nucleotides are replaced later with DNA nucleotides by DNA polymerase.
16
Q
Details of DNA Replication
A
- Helicase unwinds DNA, producing replication fork. Single-strand binding proteins prevent single strands of DNA from recombining. Topoisomerase removes twists.
- Primase initiates replication at origins or replication with short segment of RNA nucleotides, called RNA primers.
- DNA polymerase attached to RNA primers and begins elongation
- Leading complementary strand is assembled continuously
- Lagging complementary strand is assmbled in short Okazaki fragments
- Okazaki fragments joined by DNA ligase
- RNA primer replaced by DNA nucleotides
**energy for elongation comes from breaking two phosphate bonds
17
Q
Telomeres
A
- ends of eukaryotic chromosomes
- two problems:
1. when not enough template strand remains to which primase can attach
2. when last primase removed —> empty space left by removal of primer is left unfilled - Solution: enzyme TELOMERASE attached to end of template strand and extends by adding a short sequence of DNA nucleotides. DNA in extended region merely act to prevent loss of important cod’ing DNA that precedes it.
18
Q
Used to be “one gene one enzyme hypothesis” now….
A
one gene one polypeptide hypothesis
19
Q
Three steps of protein synthesis
A
- Transcription (DNA -> RNA)
- RNA processing (additions and deletions)
- Translation (RNA –>polypeptides
20
Q
3 Kinds of RNA molecules produced during transcription:
A
- Messenger RNA (mRNA). single strand RNA. template for sequencing amino acids into a polypeptide. A triplet of 3 adjacent nucleotides = codon. 64 possible codon, only 20 amino acids. Three of the codons are STOP codons, so only 61 actually code for amino acids.
- Transfer RNA (tRNA). short RNA molecule (consisting of about 80 nucleotides. transport amino acids to proper place on mRNA template. A portion is the anticodon which base pairs with the codon of mRNA. Wobble –> exact base-pairing not required for third nucleotide –> about 45 different tRNA’s base-pair with 61 codons that code for amino acids.
- Ribosomal RNA (rRNA). building blocks of ribosomes. Within nucleolus, various proteins imported from cytoplasm are assembled with rRNA to form large and small ribosome subunits –> together form RIBOSOME (3 binding sites, A,P,E)
21
Q
Transcription
A
Initiation –> Elongation –> Termination
- Initiation. RNA polymerase attached to promoter region of DNA and unzip the DNA (TATA Box)
- Elongation occurs as RNA polymerase unzips DNA and assembles RNA nucleotides using one strand of the DNA as template. As in DNA replication, elongation of RNA occurs in 5’ –> 3’. In contrast to DNA replication, new nucleotides are RNA and only one DNA strand is transcribed
- Termination occurs when RNA polymerase reaches a special sequence of nucleotides that serve as a termination point. In eukaryotes, often DNA sequence AAAAAA
22
Q
mRNA Processing
A
- Before mRNA leaves nucleus, some alterations:
1. 5’ cap (-p-p-p-G-5’) added to 5’ end of mRNA. 5’ cap is a guanine nucleotide with two additional phosphate groups, forming GTP (same way ATP is adenine with two additional phosphates). Capping provides stability and point of attachment for small subunit of ribosome.
- Poly-A tail (-AAAAA-3’) is attached to 3’ end of mRNA. tail consists of about 200 adenine nucleotides. Provides stability and appears to control movement of mRNA across nuclear envelope.
- RNA splicing. Before mRNA moves to cytoplasm, small nuclear ribonucleoproteins, or snRNP’s, delete introns and splice the exons.
- Alternative splicing. allows different mRNA’s to be produced from same RNA transcript. By selectively removing different parts of an RNA transcript, different mRNA’s can be produced, each coding for a different protein product.
23
Q
Translation
A
- after transcription, mRNA, tRNA and ribosomal subunits are transported across the nuclear envelope and into the cytoplasm.
- in cytoplasm, amino acids attach to 3’ end of tRNA’s, forming aminoacyl-tRNA.,. reaction requires energy from ATP and enzyme specific to each tRNA
- As in transcription, 3 steps to translation: Initiation, elongation, and termination. energy for translation is provided by several GTP molecules.
24
Q
Translation Steps:
A
- Initiation begins when small ribosomal subunits attaches to a special region near 5’ end of mRNA.
- A tRNA (w/ anticodon UAC) carrying amino acid methionine attaches to mRNA at start codon AUG.
- Large ribosomal subunit attaches to mRNA, forming a complete ribosome with the tRNA (bearing methionine) occupying P site.
- Elongation begins when next tRNA (bearing amino acid) binds to A site of ribosome. Then, methionine is removed from first tRNA and attached to amino acid on newly arrived tRNA.
- first tRNA, no longer carrying amino acid is released. After release, can again bind its specific AA.
- remaining tRNA (together w/ bound mRNA) move from A site to P site (translocation). Now A site is unoccupied w/ a new codon exposed. Analogous to ribosome moving over one codon.
- new tRNA carrying a new amino acid enters A site. The two AA on tRNA in P site are transferred to the new amino acid, forming a chain.
- tRNA in P site is released, and subsequent steps repeated.
- Termination occurs when ribosome encounters one of three stop codons. At termination, the completed polypeptide, the last tRNA, and the two ribosomal subunits are released.
25
Post Translation:
- once polypeptide is completed, interactions among amino acids give it its secondary and tertiary structures.
- Subsequent processing by ER or Golgi body may make final modifications before protein functions as a structural element or as an enzyme.
26
Mutations
- DNA replication is not perfect...mutations happen
- a mutation is any sequence of nucleotides in a DNA molecule that does not exactly match the original DNA molecule from which it was copied.
27
Point Mutation
- single nucleotide error. include the following:
1. substitution: incorrect nucleotide in place of correct
2. deletion: a nucleotide is omitted from nucleotide sequence
3. Insertion: a nucleotide is added to nucleotide sequence
4. frameshift: result of a nucleotide deletion or insertion. If a frameshift mutation occurs in DNA segemtn that codes for mRNA, all codons follwong transcribed mutation will change.
28
Result of Single Mutations:
1. Silent mutation. Code for same amino acid. ex. third nucleotide of codon --> wobble
2. missense mutation. new codon codes for a new amino acid. effect may be minor or not. Hemoglobin protein that causes sicle-cell disease is caused by a missense mutation.
3. nonsense mutation. new codon codes for a stop codon.
29
Mutagens
- radiation or chemicals that cause mutations
30
Carcinogens
- mutagens that activate uncontrolled cell growth (cancer)
31
Various mechanisms to repair replication errors:
1. Proofreading. DNA polymerse proofreads and corrects error if there is one.
2. Mismatch repair enzymes repair errors that escape the proofreading ability of DNA polymerase.
3. Excision repair enzymes remove nucleotide damaged by mutagens.
32
DNA Organization
- In eukaryotes: DNA packaged with proteins to form matrix called chromatin. DNA is coiled around bundles of eight or nine histone proteins to form DNA-histone complexes called nucleosomes.
- During cell division, DNA is compactly organized into chromosoems. When cell isn't dividing, DNA is arranged as either of 2 types of chromatin:
1. Euchromatin: DNA loosely bound to nucleosomes. DNA in these regions is actively being transcribed.
2. Heterochromatin: areas where nucleosomes are more tightly compacted and where DNA is inactive. Becaus eof condensed arrangement, heterochromatin stains darker than euchromatin.
33
Transposons
- AKA jumping genes
- can move to new location on same chromosome or to a different chromosome.
- whenever they are inserted, transposons have the effect of a mutation. They may change the expression of a gene, turn on or turn off its expression, or have no effect at all.
34
Molecular Genetics of Viruses
- viruses are parasites of cells. A typical virus penetrates cell, commandeers its metabolic machinery, assembles new viruses that are copies, and leaves to infect other cells. Host cell usually destroyed in process.
35
Bacteriophages
- or just phages
| - viruses that attack only bacteria
36
Structures of Viruses
1. nucleic acid. RNA or DNA but not both. double or single stranded.
2. Capsid or Protein coat. encloses the nucleic acid. Identical protein subunits, called capsomeres, assemble to form the capsid.
3. Envelope. surrounds capsid of some viruses. Envelopes incorporate phospholipids and proteins obtained from cell membrane to host.
37
Two cycles of replication of viruses:
1. Lytic cycle. Virus enters, uses host machinery to make viral nucleic acids and proteins. assemble to new viruses, leave to attack other cells, destroying the host in the process. Some variations
a. most DNA viruses --> DNA is replicated to form new viral DNA ---> transcribed --> viral proteins --> assemble to new viruses
b. RNA viruses, RNA serves as mRNA or template to make mRNA. The mRNA is translated to make proteins --> proteins assembled w/ RNA to make new viruses.
c. Retroviruses: ssRNA viruses that use an enzyme called reverse transcriptase to make DNA complement to their RNA. The DNA complement can be transcribed immediately to manufacture mRNA, or begin lysogenic cycle by becoming incorporated in DNA of host. HIV, the cause of AIDS is a retrovirus.
2. Lysogenic cycle. Viral DNA temporarily incorporated into DNA of host cell. Virus in this dormant state is called provirus (or, if a bacteriophage, a prophage). Virus remains inactive until some trigger (ex. radiation)
38
Molecular Genetics of Bacteria
- bacteia = prokaryotes
- don't contain nucleus or specialized organelles
- priamry genetic material: single circular DNA molecule
- bacterial chromosme often called a naked chromosome because it lacks histones and other proteins
- bacterial cell reproduces by binary fission --> chromosomes replicate and cell divides in two. Spindle apparaturs, microtubules, and centrioles are lacking since there is no nucleus to divide.
39
Plasmid
- short, circular DNA molecules outside chromosome found in bacteria.
- carry beneficial genes but not normally essential
- replicate independently of chromosome.
- some plasmids, called episomes, can become incorporated into bacterial chromosme.
40
Ways in which genetic variation is introduced into genome of bacteria:
1. Conjugation: DNA exchange between bacteria. Donor bacterium produces tube, or pilus that connect to recipient bacterium. Chromosomal or plasmid DNA can be sent.
a. F plasmid: enable bacterium to produe pili
b. R plasmid: provide resistance against antibiotics.
2. Transduction: new DNA introduced into genome of bacterium by a virus. When virus assembles during lytic cycle, it is sometimes assembled with some bacterial DNA --> when this virus infects another cell, the bacterial DNA it delivers might recombine with the resident DNA.
3. Transformation: bacteria absorb DNA from surroundings and incorporate into their genome.
41
Regulation of Gene Expression
- every cell in human body contains exactly same DNA sequence. Cells become different by regulation of gene expression through transcription of only selected gene.
- ex. operon
42
Operon
- unit of DNA among prokaryotes that controls Transcription of a gene.
- has the following components:
1. Promoter region: sequence of DNA to which RNA polymerase attaches to begin transcription.
2. Operator: can block action of RNA polymerase if occupied by a repressor protein.
3. Structural gene: DNA sequence that code for several related enzymes that direct production of some particular end product.
4. Regulatory gene: laying outside operon. Produces repressor/activator proteins, substances that occupy operator region and block/activate action of RNA polymerase (transcription).
43
Two kinds of operons in bacterium E. coli?
- lac operon
- trp operon
- common bacterium in digestive tracts of humans
44
Lac operon
- E.coli
- controls breakdown of lactose: regulatory gene produces repressor that binds operator and stops RNA polymerse from transcribing the structural genes that code for enzymes that control uptake and breakdown of lactose. When lactose is available --> lactose binds repressor and make it inactive ---> repressor inactive -------> RNA polymerase transcribes genes that code for enzymes that breakdown lactose.
**since a substance, lactose is required to induce (turn on) the operon, the enzyme that the operon produces are said to be inducible enzymes.
45
Trp Operon
- E. coli
- produces enzymes for synthesis of amino acid tryptophan. Regualtory gene produces an inactive repressor ---> RNA polymerase able to bind and transcribe structural genes needed to produce enzymes that synthesize tryptophan. If tryptophan is already available to E. coli ---> typtophan (corepressor) react with inactive repressor and make it active --> active repressor binds operator and prevent transcription
** since structural genes stop producing enzymes only in presence of active repressor, they are called repressible enzymes.
46
Mechanisms of Gene Expression in Eukaryotic Cells:
1. Regulatory proteins: repressors and activators operate similarly to those in prokaryotes, influencing how RNA polymerase will attach to a promoter region. In many cases, numerous activator act together to influence transcription
2. Nucleosome Packaging transcription of DNA. DNA segments tightly packed by methylation of histones make transcription more difficult. In contrast, acetylation of histones allows uncoiling and transcription of DNA.
3. RNA interference. Short interference RNAs (siRNAs) block mRNA transcription or translation or degrade existing mRNA. Essentially, base-pair with complementary DNA or mRNA and preventing transcription/translation. In other cases, siRNAs combine with enzymes to degrade existing mRNAs with complementary sequences.
47
Recombinanat DNA
- contains DNA segments or genes from different sources. DNA from one part of a DNA molecue to another; from one chromosome to another, or from one organisms to another.
- transfer of DNA can occur naturally or through viral transduction, bacterial conjugation, or transposons, or artificially through reombinant DNA technology.
- Crossing over during prophase I of meiosis produces recombinant chromosomes.
48
Recombinant DNA Technology
- use restriciton enzymes to cut up DNA. Restriction enzymes obtained from bacteria that manufacture these enzyme to combat viruses.
- Restriction enzyme are very specific. The cut acorss ds DNA is usually staggered --> sticky ends
- To insert a foreign DNA segment into another cell the fragment is first introduced into another DNA molecule, or Vector. Plasmids are commonly used because they can subsequently be introduced into bacteria by transformation. Plasmid treated w/ same restriction enzyme used to create the foreign DNA fragment producing same sticky ends. Mixing foreign DNA with cut plasmid allows base-pairing at sticky-ends. Application of DNA ligase stablizes the attachments. Recombinant plasmid is then introduced into a bacterium by transformation.
***By following this procedure, the human gene for insulin has been inserted into E. coli. Transformed E. coli produces insulin which is isolated and used to treat diabetes.
49
Gel Electrophoresis
- Restriction fragments can be separated by gel electrophoresis.
- DNA fragments of different lengths are separated as they diffuse through a gelatinous material under influence of an electric field. Since DNA is negatively charged (phosphate groups), it moves toward the positive electrode. Shorter fragments migrate further than heavier fragments.
- Gel electrophoresis is often used to compare DNA fragments of closely related specis in an effort to determine evolutionary relationships.
50
When restriction fragments between individuals of same species are compared, the fragments differ in length because of.....
- polymorphisms
- Polymorphisms are slight differences in DNA sequences. These fragments are called restriction fragment length polymorphisms, or RFLPs,
- In DNA fingerprinting, RFLPs produced from DNA left at a crime scene are compared to RFLPS from DNA of suspects. Areas of human genome that are particularly polymorphic contain SHORT TANDEM REPEATS (STRs).
- STRs are short sequences of nucleotides (2 to 5) that repeat multiple times, with number of repeats varying markedly among individuals.
51
Complementary DNA
- when foreign genes are inserted into genome of a bacterium with recombinant DNA technology, introns often prevent their transcription. To avoid this problem, the DNA is obtained directly from mRNA of desired polypeptide using reverse transcriptase (from retroviruses)
- DNA obtained in this manner is called complementary DNA, or cDNA. Lacks introns that suppress transcription.
52
Polymerase Chain Reaction (PCR)
- instead of using bacterium to clone DNA fragments, fragments can be copied millions of times using DNA polymerase directly.
- This method, called PCR, uses synthetic primers that initiate replication at specific nucleotide sequecnes.
