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Biochem > DNA & RNA > Flashcards

DNA & RNA Flashcards

1
Q

DNA metabolism

A
  • DNA provides stable storage of genetic information, BUT, the structure is far from static:
    o New copy of DNA is synthesized before each cell division.
    o Errors that arise in DNA synthesis are constantly repaired
    o Segments of DNA are rearranged either within a chromosome or between two DNA molecules (recombination), giving offspring a novel DNA.
  • DNA metabolism consists of tightly regulated processes to achieve these tasks.
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2
Q

DNA replication properties

A
  • Three fundamental rules of replication:
    1. Replication is semi-conservative
    2. Replication begins at an origin and proceeds (usually) bi-directionally (esp in bacterial cells)
    3. Synthesis of new DNA occurs in the 5’–>3’ direction and is semi-discontinuous

Synthesis of new DNA 5’ –> 3’: synthesis of the new strand occurs in this direction. Reading the DNA you will be in 3’ –> 5’ – because it is complementary… new nucleotides added to 3’ end of the strand

Semi-discontinuous: one strand = continuous, other strand = discontinuous

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3
Q

semiconservative replication

A
  • Synthesis proceeds in direction of 5’ –> 3’
  • Synthesis always occurs by addition of new nucleotides to the 3’ end (3’-OH).
  • The leading strand is made continuously as the replication fork advances.
  • The lagging strand is made discontinuously in short pieces (Okazaki fragments) that are later joined together.
  • Parent strand separted, two new daughter strands created, each bind to a parent strand. So new DNA made up of 1x daughter and 1x parent strand.
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4
Q

leading and lagging strand synthesis

A
  • synthesized 5’ –> 3’ direction but the template is read 3’ –> 5’ direction
  • The leading strand is continuously synthesized in the direction taken by the replication fork.
  • The other strand, the lagging strand, is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves.
  • The Okazaki fragments are spliced together by DNA ligase.
  • In bacteria, Okazaki fragments are 1,000 to 2,000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long.
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5
Q

DNA degradation

A
  • Nucleases degrade nucleic acids.
    o Specifically, DNases degrade only DNA; RNases degrade only RNA.
  • Exonucleases cleave bonds that remove nucleotides from the ends of DNA.
  • Endonucleases cleave bonds within a DNA sequence.
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6
Q

DNA synthesis

A
  • Synthesised by DNA polymerases
  • First (DNA polymerase I) discovered by Arthur Kornberg in E. coli
  • E. coli contains at least four other DNA polymerases.
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7
Q

DNA elongation chemistry

A
  • Parental DNA strand serves as a template.
  • Nucleoside triphosphates serve as substrates in strand synthesis.
  • The nucleophilic OH group at the 3’ end of the growing chain attacks the α-phosphate of the incoming trinucleotide.
    o This 3’-OH is REQUIRED.
    o The 3’-OH is made a more powerful nucleophile by nearby Mg2+ ions.
  • Pyrophosphate (made of the γ and β phosphates) is a good leaving group.
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8
Q

mechanism of DNA polymerases

A

The Mg2+ ion depicted at the top facilitates attack of the 3’-hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate; the other Mg2+ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state

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9
Q

primer for DNA polymerase

A
  • Primer = short strand complementary to the template
    o contains a 3’-OH to begin the first DNA polymerase-catalyzed reaction
    o can be made of DNA or RNA (more common)
  • Nucleotide is added and then DNA polymerase moves along the strand = translocation
  • Diagram shows primer and where new DNA strand is being added
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10
Q

DNA polymerase

A
  • Enzyme has a pocket with two regions:
    o insertion site: incoming nucleotide binds
    o Post-insertion site: where newly made base pair resides when the polymerase moves on
  • DNA polymerase active site excludes base pairs with incorrect geometry
    o wrong base once in 1/104–1/105 times.
    o Repair mechanisms fix these errors.
    o Incorrect base pairs: due to wrong base, cannot fit into active site
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11
Q

base pair geometry

A

The standard A=T and G≡C base pairs have very similar geometries, and an active site sized to fit one will generally accommodate the other. (b) The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase.

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12
Q

errors during synthesis: exonuclease activity

A
  • Errors during synthesis are corrected by 3’ –> 5’ Exonuclease Activity
  • ~All DNA polymerases have an additional activity.
  • 3’ –> 5’-exonuclease activity “proofreads” synthesis for mismatched base pair
  • Translocation of enzyme to next position is inhibited until the enzyme can remove the incorrect nucleotide
    If introduces incorrect base pair, it will introducing a mutation, so want to avoid
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13
Q

DNA polymerase I

A
  • Has 5’ –> 3’-Exonuclease Activity
  • In addition to the 3’ –> 5’-exonuclease activity
  • Moves ahead of the enzyme, hydrolyzes things in its path
  • Performs nick translation―a strand break moves along with enzyme
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14
Q

DNA polymerase III

A
  • Complex structure with 10 types of subunits

- Responsible for DNA Replication

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15
Q

DNA replication

A 
- Many conserved principles between prokaryote and eukaryotes
Three stages:
1. Initiation
2. Elongation
3. Termination
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16
Q

requirements for E. coli DNA replication

A
  • E. coli requires over 20 enzymes and proteins
  • The set is called the replisome. (the 20 enzymes and proteins in addition to DNA polymerase)
  • Includes/key parts:
    o Topoisomerases (gyrase) (relieve the stress caused by unwinding)
    o helicases (use ATP to unwind DNA strands)
    o DNA-binding proteins to stabilize separated strands
    o primases to make RNA primers
    o DNA ligases to seal nicks between successive nucleotides on the same strand (i.e., Okazaki fragments)
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17
Q

stage 1: initiation of replication (E. coli)

A
  • Begins at the oriC site – site of origin (245 bp in length)
  • Requires at least 10 different proteins
    o Includes helicase & gyrase
  • Goal: open the helix, form pre-priming complex
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18
Q

stage 2: elongation (leading strand)

A
  • Straightforward approach
  • Primase makes RNA primer (10–60nt).
  • DNA Pol III adds nucleotides to the 3’ end of the strand.
  • ~1,000–2,000 nt/sec
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19
Q

lagging strand synthesis

A
  • Synthesis of Okazaki fragments
  • At intervals, primase synthesizes an RNA primer for a new Okazaki fragment. Note that if we consider the two template strands as lying side by side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. Each primer is extended by DNA polymerase III. DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment. A new primer is synthesized near the replication fork to begin the process again
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20
Q

transition between Okazaki fragments

A
  • Core subunits of DNA Pol III dissociate from one  clamp and bind to a new one
  • RNA primer is removed by DNA Pol I or RNase H1
  • DNA Pol I fills in the gap
  • DNA ligase seals the backbone
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21
Q

final step in synthesis of lagging strand

A
  • RNA primers in the lagging strand are removed by the 5’ –> 3’ exonuclease activity of DNA polymerase I and are replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. The role of ATP or NAD+ is shown to right.
  • High energy requiring process
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22
Q

DNA ligase

A
  • makes a bond between a 3’-OH and a 5’-PO4
  • 5’-PO4 must be activated by attachment of AMP.
  • 3’-OH nucleophile attacks this phosphate, displacing AMP.
  • Forms the Phosphodiester bond, key in DNA and RNA
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23
Q

stage 3: termination

A
  • In E. Coli the Replication forks meet at a region with 20-bp (Ter) termination sequences
  • Causes replication fork to stop
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24
Q

replication in eukaryotes

A
  • More complex than bacteria
  • Occurs more slowly than E. coli does
    o (~1/20th the rate seen in E. coli)
  • However, compensated by multiple origins (every 30–300 kb). Eukaryotic DNA much bigger, to overcome time constraints, replication occurs at multiple sites.
  • Uses multiple DNA Polymerases
    o DNA Pol α - polymerase/primase activity
    o DNA Pol δ (lagging strand)
    o DNA Pol ε (leading strand)
  • Termination occurs at the Telomeres
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25
Q

DNA repair and mutations

A
  • Chemical reactions and some physical processes constantly damage genomic DNA.
    o The majority are corrected using the undamaged strand as a template.
    o Some base changes escape repair, and an incorrect base serve as a template in replication.
    o The daughter DNA carries a changed sequence in both strands.
  • Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens.
  • There are thousands of lesions/day (unrepaired DNA damage) but only 1/1,000 become a mutation, thanks to DNA repair.
  • The human genome contains genes for > 130 repair proteins.
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26
Q

DNA lesions

A
  • Lesion = DNA damage
  • If unrepaired, lesion becomes mutation
    o Mutations can be substitutions (point mutations), deletions, additions
  • Silent mutation―has ~no effect on gene function or affects a nonessential region of the DNA
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27
Q

types of DNA damage

A
  • Mismatches arise from occasional incorporation of incorrect nucleotides.
  • Abnormal bases arise from spontaneous deamination, chemical alkylation, or exposure to free radicals.
  • Pyrimidine dimers form when DNA is exposed to UV light.
  • Backbone lesions occur from exposure to ionizing radiation and free radicals.
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28
Q

functions of nucleotides and nucleic acid (NA)

A
  • Nucleic acids are polymers of nucleotides used for:
    o storage of genetic info (DNA)
    o transmission of genetic info (mRNA)
    o processing of genetic information (ribozymes)
    o protein synthesis (tRNA and rRNA)
  • Nucleotides are also used in the monomer form for cellular functions:
    o energy for metabolism (ATP)
    o enzyme cofactors (NAD+)
    o signal transduction (cAMP)
29
Q

nucleotides and nucleosides

A 
- Nucleotide consists of
o nitrogeneous base
o pentose sugar
o phosphate
- Nucleoside consists of 
o nitrogeneous base
o pentose sugar
- Carbon AND nitrogen atoms on the nitrogenous base are numbered in cyclic format. 
- Carbons of the pentose are designated N’ to alleviate confusion.
30
Q

nitrogenous bases

A
  • Derivatives of pyrimidine or purine
  • Nitrogen-containing heteroaromatic molecules
  • Planar or almost planar structures
  • Absorb UV light around 250–270 nm

Pyrimidine and Purine Bases

  • Cytosine, adenine, and guanine are found in both DNA and RNA.
  • Thymine is found only in DNA.
  • Uracil is found only in RNA.
  • All are good H-bond donors and acceptors.
  • Neutral molecules at pH 7
  • Pyrimidine = single ring
    o Cytosine, thymine/uracil
  • Purine = Double ring structure
    o Adenine, guanine
31
Q

modified nucleosides in DNA

A
  • Modification is done after DNA synthesis.
  • 5-Methylcytosine is common in eukaryotes and is also found in bacteria.
  • Epigenetic marker:
    o way to mark own DNA so that cells can degrade foreign DNA (prokaryotes)
    o way to mark which genes should be active (eukaryotes)
    o Could the environment turn genes on and off in an inheritable manner? Eg is possibly a product of environment.
  • Protective mechanism
32
Q

polynucleotides

A
  • Covalent bonds are formed via phosphodiester linkages: negatively charged backbone (due to phosphate attachment)
  • DNA backbone is fairly stable: Eg DNA has been extracted from remains of mammoths
    o Stable due to H bonds and phosphodiester bonds
    o Hydrolysis accelerated by enzymes (DNAse)
  • RNA backbone is unstable.
    o In water, RNA lasts for a few years.
    o In cells, mRNA is degraded in a few hours.
    o Unstable due to hydroxyl groups instead of H bonds; more susceptible to hydroylysis
  • Linear polymers
    o no branching or cross-links
  • Directionality
    o The 5’ end is different from the 3’ end.
    o We read the sequence from 5’ to 3’.
33
Q

hydrolysis of RNA

A
  • RNA is unstable under alkaline conditions.
  • Hydrolysis is also catalyzed by enzymes (RNase).
  • RNase enzymes are abundant around us.
    o S-RNase in plants prevents inbreeding.
    o RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors.
    o Dicer is an enzyme that cleaves double-stranded RNA into oligonucleotides: protection from viral genomes & RNA interference technology
34
Q

phosphodiester linkages

A
  • In the covalent backbone of DNA and RNA
  • The phosphodiester bonds link successive nucleotide units. The backbone of alternating pentose and phosphate groups in both types of nucleic acid is highly polar. The 5’ and 3’ ends of the macromolecule may be free or may have an attached phosphoryl group
35
Q

hydrogen bonding interactions

A
  • Two bases can hydrogen bond to form a base pair.
  • For monomers, a large number of base pairs is possible.
  • In polynucleotide, only a few possibilities exist.
  • Watson-Crick base pairs predominate in double-stranded DNA.
  • Purine pairs with pyrimidine.
  • A pairs with T (2 H bonds) [A=H]
  • C pairs with G (3 H bonds) [C=G]
36
Q

complementary structure of DNA strands

A
  • Two chains differ in sequence
    o (sequence is read from 5’ to 3’).
  • Two chains are complementary.
  • Two chains run antiparallel.
37
Q

replication of genetic code

A
  • Strand separation occurs first.
  • Each strand is a template for the synthesis of a new strand.
  • Synthesis is catalyzed by the enzymes - DNA polymerases.
  • A newly made DNA molecule has one daughter strand and one parent strand.

Parent strand separated, and each individua strands are copied. Newly made DNA molecule made up of 1xdaughter and 1xparent strand

38
Q

mRNA

A
  • Messenger RNA: mRNA code carrier for the sequence of proteins
  • Is synthesized using DNA template as a single strand
  • Contains ribose instead of deoxy-ribose
  • Contains uracil instead of thymine
  • One mRNA may code for more than one protein
  • RNA Molecule Structure complex structure relies on HYDROGEN BONDING
  • Together with transfer RNA (tRNA), transfers genetic information from DNA to proteins
  • mRNA: copy of gene
  • tRNA: transfers AA to allow protein synthesis
  • rRNA: reads mRNA
39
Q

hairpins

A

mRNA contains palindromic sequences that can form hairpins and cruciform: Palindromic DNA (or RNA) sequences can form alternative structures with intrastrand base pairing. When only a single DNA (or RNA) strand is involved, the structure is called a hairpin bend – nucleotides lined up against each other, perfect complementarity. Good thing, contributes to tRNA structure. mRNA has hairpins = more stable, degrades slower.

40
Q

DNA denaturation

A

Heating and then cooling of DNA
- Covalent bonds remain intact.
o Genetic code remains intact.

  • Hydrogen bonds are broken: Two strands separate.
  • Base stacking is lost: UV absorbance increases.
  • Denaturation can be induced by high temperature or change in pH.
  • Denaturation may be reversible: annealing.
  • Some degradation does/can occur
  • not uniform: AT-rich regions melt at a lower temperature than GC-rich regions.
  • Remember – A=T 2H bonds C=G 3H bonds, so C=G requires more energy to break
41
Q

factors affecting DNA denaturation

A
  • The midpoint of melting (Tm): Tm depends on base composition.
    o High CG increases Tm because has 3x H bonds. AT lower Tm due to 2x H bonds.
  • Tm depends on DNA length.
    o Longer DNA has higher Tm.
    o It is important for short DNA.
  • Tm depends on pH and ionic strength.
    o High salt increases Tm.
42
Q

function of nucleotides as an energy source

A

General structure of the nucleoside 5’-mono-, di-, and triphosphates (NMPs, NDPs, and NTPs) and their standard abbreviations. In the deoxyribonucleoside phosphates (dNMPs, dNDPs, and dNTPs), the pentose is 2’-deoxy-D-ribose. Some cells can use GTP and CTP as energy, over ATP

43
Q

function of nucleotides

A
  • Coenzyme: as a component of their structure is a nucleotide
    o Eg Acetyl-CoA or coenzymeA
  • Regulatory molecule
    o Cyclic AMP; cAMP
44
Q

overview of RNA function

A

Ribonucleic acids play three well-understood roles in living cells:

  1. Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell.
  2. Transfer RNAs match their anticodon to the mRNA while carrying a specific amino acid used for protein synthesis.
  3. Ribosomal RNAs are constituents of the large and small ribosomal subunits.

Ribonucleic acids play several less-understood functions in eukaryotic cells:

  1. MicroRNAs regulate the expression of genes, possibly via binding to specific nucleotide sequences.
  2. Ribozymes are catalytic RNA molecules that act as enzymes.
    - Ribonucleic acids act as genomic material in viruses.
45
Q

RNA metabolism overview

A
  • Transcribed from DNA: Transcription is tightly regulated in order to control the concentration of each protein that is present.
  • Ribozymes: Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors such as the group I introns. Also play a roll in splicing.
  • Processing of mRNAs
    1. Splicing - elimination of introns; joining of exons
    2. Poly-adenylation of the 3’ end
    3. Capping the 5’ end
46
Q

transcription in E. coli

A
  • The nucleoside triphosphates add to the the 3’ end of the growing RNA strand (therefore 5’ –> 3’ direction) (RNA strand synthesized same direction as in DNA)
  • The growing chain is complementary to the template strand in DNA.
  • The synthesis is catalyzed by the enzyme RNA polymerase (requires 2 Mg2+ ions for catalysis)
  • RNA polymerase unwinds ~17bp of DNA and covers about 35 bp of DNA.
47
Q

feature of transcription

A
  • RNA polymerase binds to a sequence called promoter to begin transcription (no primer required)
  • The growing end of new RNA temporarily base-pairs with DNA template for ~8 bp (inserts 50-90nt/sec)
  • The DNA duplex/helix unwinds, forming “bubble” of separated strands, ~17 bp in length
  • The RNA Pol generates positive supercoils ahead, later relieved by topoisomerases (Gyrases) that stabilise and prevent too tightly coiling
48
Q

transcription bubble

A

Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. About 17 bp of DNA are unwound at any given time. RNA polymerase and the transcription bubble move from left to right along the DNA as shown, facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. As the DNA is rewound, the RNA-DNA hybrid is displaced, and the RNA strand is extruded.

49
Q

tempalte vs coding strand

A
  • DNA template strand – serves as template for RNA polymerase (ie. The strand being copied into RNA)
  • DNA coding strand – the non-template strand; has the same sequence as the RNA transcript (ie. codes for the protein)
  • **Regulatory sequences are contained on the coding strand sequence

The two complementary strands of DNA are defined by their function in transcription. The RNA transcript is synthesized on the template strand and is identical in sequence (with U in place of T) to the nontemplate strand, or coding strand.

RNA transcript matches coding strand, but T substituted with U

50
Q

RNA polymerase lack of proofreading capability

A
  • RNA polymerase holoenzyme has five core subunits of α2ββ’ω plus a sixth called σ
  • RNA Pol lacks 3’–> 5’-exonuclease, so it has a high error rate of 1/104–1/105.
  • RNA binds to promoter regions to initiate transcription.
  • More susceptible to copying error
51
Q

common features of promoters in E. coli

A
  • There are two consensus sequences at −10 (TATAAT) and −35 (TTGACA) for σ subunit binding: called TATA sequences (found in both eukaryotes and prokaryotes)
  • An A-T−rich upstream promoter element between −40 and −60 binds the α subunit.
  • A-T−rich sequences promote strand separation.
  • These sequences govern efficacy of RNA Pol binding and therefore affect gene-expression level.
  • Nucleotides before the first nucleotide of the RNA molecule are considered “upstream” and given negative values.
52
Q

stages of transcription

A

Stage 1. Initiation of Transcription

  • RNA Pol binds to promoter with the factor bound: It creates a closed complex (DNA is not unwound).
  • An open complex forms: The region from ~−10 to ~+2 unwinds.
  • RNA Pol moves away from the promoter: σ is replaced by the protein NusA.

Stage 2. Elongation of transcription

  • RNA Pol binds to triphosphate nucleosides and generates the RNA transcript.
  • RNA pol encapsulates DNA, and separates it, adds nucleotides in channel, and has an exit channel to direct RNA away from DNA

Stage 3. Termination of Transcription

  • Two Types of Termination
    1. ρ independent: characterized by three U’s near the 3’, form a hairpin 15−20 nt before the 3’ end, makes the RNA Pol dissociate
    2. ρ-dependent: protein processes until termination (rut) site reached
53
Q

transcription regulation

A
  • Transcription is energy-intensive, so logical to regulate gene production here
  • Regulation is achieved in many ways
  • One way is to regulate the affinity of RNA polymerase for a promoter.
    1. promoter sequence – deviating from the consensus sequence
    2. activator proteins – CRP
    3. repressor proteins – block binding sites
  • cAMP receptor protein (CRP) is a regulatory protein in bacteria. CRP protein binds cAMP, which causes a conformational change that allows CRP to bind tightly to a specific DNA site in the promoters of the genes it controls.
  • Logical place to regulate gene expression, as it is where DNA is produced, can turn on or off production of an important protein, depending on cell requirements. Cell can regulate expression of different proteins, based on transcription of specific genes.
54
Q

eukaryote RNA polymerases

A
  • RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs)
  • RNA polymerase II synthesizes mRNA.
    1. very fast (500–1000 nucleotides/sec)
    2. can recognize thousands of promoters
  • RNA polymerase III makes tRNAs
  • Plants have RNA polymerase IV – synthesizes small interfering RNAs (siRNAs)
  • Mitochondria have their own RNA polymerase
55
Q

features of promoters recognised by eukaryotic RNA polymerase II

A

Features of Some Promoters Recognized by Eukaryotic RNA Polymerase II

  • Consensus sequence TATA(A/T)A(A/T)(A/G) ~−30
  • Inr sequence (Initiator) ~+1
  • Specific regulatory sequences farther upstream
  • TATA box is a recognition site, if red box is gene, various upstream regulatory sequences, where a binding protein might bind to activate and RNA polymerase binding, or a repressor protein changes to prevent
56
Q

assembly of RNA polymerase II at promoter

A
  • Initiated by TATA-binding protein (TBP) with the promoter
  • TBP is part of multi-subunit complex Transcription Factor II (TFII)
    TF in general: proteins bind to DNA and help mRNA transcription to occur. TFII helps put mRNA over start codon of gene
  • helps position RNA polymerase II over the transcription start site of the gene
  • Also contains Helicase subunit to unwind DNA at the promoter.
  • Also, Kinase activity phosphorylates RNA Pol II at the CTD (carboxy-terminal domain), changing the conformation and enabling transcription (good way to regulate an enzyme)
57
Q

elongation and termination

A
  • After 60-70nt, TFII is released
  • Elongation factors bound to RNA Pol II enhance processing and coordinate posttranslational modifications.
  • For termination, Pol II is dephosphorylated.
58
Q

processing of mRNA overview

A
  • Dozens of proteins coordinate with each other and with proteins involved in RNA transport to ribosomes (out of cytosol)
  • An unprocessed, newly synthesized RNA molecule is referred to as a primary transcript.
  • Processing includes:
    1. adding a 5’-cap
    2. adding a 3’-poly(A) tail
    3. splicing out introns and rejoining any exons for a continuous sequence
    4. degradation
59
Q

processing step 1: the 5’ cap

A
  • 7-methylguanosine links to 5’-end
  • formed with a molecule of GTP
  • Protects RNA from nucleases
  • Forms a binding site for ribosome
60
Q

processing step 2: addition of the 3’-Poly(A) tail

A
  • Serves as binding site on mRNA
  • protects mRNA from degradation
  • RNA Pol II synthesizes RNA beyond the cleavage signal sequence
    1. Endonuclease cleaves RNA 10−30 nt downstream to highly conserved AAUAA.
    2. Polyadenylate polymerase synthesizes 80−250 nt of A.
61
Q

processing step 3: introns are present in most genes

A
  • Most genes in vertebrates, and some in yeast, a few bacteria have introns.
  • Exons are usually <1,000 bp in length.
  • Introns are 50−700,000 bp in length.
    o 1,800bp avg
  • Some genes have dozens of introns.
    o The human genome has more than 200,000 introns spread across ~20,000 genes.
  • Introns must be removed for mature RNA (keep exons)
62
Q

four classes of introns

A
  • group 1 & 2 introns are self splicing
  • most common introns are regulated by complexes called spliceosomes
  • tRNA introns are spliced by protein-based enzymes
63
Q

spliceosome

A
  • The spliceosome is made up of snRNPs
  • “snurps” for small nuclear ribonuclear proteins - 100−200 nt long
  • GU at 5’-end and AU at 3’-end usually mark sites of splicing.

Binding of snRNPs

  • Contain regions complementary to mRNA
  • Help define the 5’-splice site.
  • Binds near the 3’-end of the intron.

Spliceosomes assembly

  • At least 50 proteins create a spliceosome.
  • ATP is required for assembly but not cleavage.
  • Some parts are attached to the CTD (carboxy-terminal domain) of RNA Pol II.
  • indicates coordination of splicing with transcription
  • requires a lot of energy
64
Q

alternative splicing

A
  • A single gene can yield different peptides depending on RNA processing.
  • Particular regions may be retained or removed, yielding different mature transcripts.
  • At least 95% of human genes are alternatively spliced.
  • Two mechanisms for the alternative processing of complex transcripts in eukaryotes.
  • Alternative splicing patterns. Two different 3’ splice sites are shown. In both mechanisms, different mature mRNAs are produced from the same primary transcript.
65
Q

processing step 4: degradation of cellular mRNAs

A
  • RNA half-life one means of gene regulation.
    1. Each gene product only needed for a unique time .
  • Half-lives vary from seconds to hours.
    1. rate of synthesis : rate of degradation
    2. steady state – rates are equal and balanced
    3. typical vertebrate mRNA ~3 hrs, (~1.5 mins for bacteria)
  • Degradation occurs via ribonucleases.
  • Hairpin structures in bacterial mRNA can extend half-life.
  • In eukaryotes, the 5’cap and 3’ poly (A) tail aid in the stability of the mRNA.
66
Q

ribozymes (RNA enzymes)

microRNAs (miRNAs)

A
  • Cleave themselves or another RNA
  • Enzymatic function
  • MicroRNAs (miRNAs):
    1. short noncoding RNAs of ~22 nucleotides
    2. bind to specific regions of mRNA to alter translation
    3. assist in cleaving the mRNAs
    4. or block the mRNA from translation
    5. About 1,500 human genes encode miRNAs and 1 or more affect the expression of MOST protein-coding genes!
    6. Synthesized from larger precursors: processed by two endoribonucleases, Drosha and Dicer
67
Q

telomeres

A
  • Structures at the ends of linear eukaryotic chromosomes
  • Have tandem repeats usually of T1-4G1-4
    o with A-C on the opposing strand
  • Can be tens of thousands of bp long in mammals
  • TG strand is longer than its complement, leaves a 3’-overhang of several hundred bases
  • Mechanism to ensure don’t lose coding part of DNA
68
Q

telomerase

A
  • Extends the ends of chromosomes
  • Telomeres are not easily replicated using DNA polymerases.
    1. Beyond an end, there is no template for an RNA primer.
    2. Chromosomes are shortened with each generation.
  • Telomerase adds telomeric sequences to solve this problem.

Biochem (17 decks)

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