Mmbio Exam #3

Front 1

RNA polymerase:

Back 1

An Enzyme that makes RNA from a DNA template.

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Core enzyme:

Back 2

Enzyme that sticks to DNA initially (no sigma factor)

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Holoenzyme:

Back 3

Core enzyme plus sigma factor, directs core enzyme to bind specifically to certain gene promoters

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Sigma factors:

Back 4

Proteins that bind to RNA polymerase (to make the holoenzyme) and these direct the polymerase to certain gene promoters

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Transcription factor:

Back 5

A molecule (usually a protein) that regulates whether or not transcription will take place or not take place

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Terminator:

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Terminator means that transcription stops here (RNA sequences that kick off RNA polymerase; here label is affixed to DNA that encodes those RNA sequences)

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Upstream:

Back 7

A term used to label gene sequences and are (- #’s) relative to base # +1

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Downstream:

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A term used to label gene sequences and are (+ #’s) relative to base # +1

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Promoter:

Back 9

A region of DNA that is nearly always directly upstream of the start site of transcription. Transcription factors and RNA polymerase bind here to prepare to initiate transcription.

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3 Phases of RNA Transcription

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Initiation: getting the process started
– Bringing the necessary proteins to the appropriate locations on the DNA, adding the first nucleotides into place

Elongation: polymerizing
– Adding onto the chain
Termination: stopping polymerization and detaching from DNA

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Differences between mechanisms of RNA transcription and DNA replication:

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- You use both strands of DNA for replication, just one for RNA transcription
- RNA transcription is much quicker than DNA replication
- Core Nucleotides: RNA transcription produces Uracil instead of Thymene
- Sequences compared to sense strand : again, RNA is exactly same (U instead of T)
- DNA polymerase is semi-discontinuous, RNA is continuous
- DNA has primase to tell it where to bind, RNA does not (has promoter region)
- DNA reaches the end to stop, RNA reaches TERMINATOR sequences

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Open reading frame (ORF):

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a DNA sequence that contains a start codon and stop codon (usually implies that a protein is produced)

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Monocistronic:

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a single mRNA that encodes a single ORF/polypeptide chain (protein)

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Polycistronic:

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a single mRNA that encodes multiple ORFs/polypeptide chains/proteins

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Operon:

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2 or more contiguous ORFs under the control of a single operator

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Operator:

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a component of a promoter that gives additional control over whether or not gene is actively transcribed

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Repressor:

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A Molecule that binds to promoter/operator in order to block access to promoter: no transcription

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Inducer:

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Inducer molecule binds to repressor, changes it’s shape, and blocks ability to bind to promoter/operator

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Inducible gene expression:

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Only turn these genes on when they are necessary (normally off)

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Repressible gene expression:

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Turn genes off when they are no longer necessary (normally on)

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Constitutive gene expression:

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Gene is always on, no matter what

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Attenuator:

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Found in the TrpL sequence or leader sequence. Functionality is in the RNA. found at the 5 prime end of the trp transcripts

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Polynucleotide phosphorylase (PNPases):

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A enzyme involved in mRNA degradation in bacteria. Mechanism is to break phosphodiester bonds by adding in a second phosphate group, works in 3’ to 5’ direction (exonuclease). You need a second enzyme to get past 3’ hairpin structures common in mRNAs, which block PNPase

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Attenuator:

Back 24

Found in the TrpL sequence or leader sequence. Functionality is in the RNA. found at the 5 prime end of the trp transcripts

Front 25

Polynucleotide phosphorylase (PNPases):

Back 25

A enzyme involved in mRNA degradation in bacteria. Mechanism is to break phosphodiester bonds by adding in a second phosphate group, works in 3’ to 5’ direction (exonuclease). You need a second enzyme to get past 3’ hairpin structures common in mRNAs, which block PNPase

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Transcription initiation:

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Basic idea is on or off. No fine-tuning.

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Transcription initation when plenty of tryptophan is present:

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1. Tryptophan binds to repressor which changes its shape and allows it to bind to trpP and trpO sequence of DNA.
2. This blocks transcription thus causing no tryptophan to be produced
3. This reduces transcription by 70-fold

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Transcription initiaion when low amounts of tryptophan are detected:

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1. Tryptophan does not bind to repressor thus causing no change in shape which prevents it binding to the trpP and trpO sequence of DNA.
2. Transcription is not blocked and tryptophan is produced

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Transcription elongation/termination:

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happens when you want intermediate levels
KEY: Transcription has already begun, rate of ribosome movement on mRNA affects the formation of secondary RNA structures (Secondary structures are regions of double stranded RNA which can act as terminator sequences-Stem loops), this type of mechanism only happens in Prokaryotes (no nucleus).

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Two categories of bacterial enzymes used to degrade mRNA's:

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Ribonucleases (RNases)
Polynucleotide phosphorylases (PNPases)

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Ribonucleases (RNases)

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cleave phosphodiester bonds using water (hydrolytic cleavage), endonuclease cut in the middle and exonuclease cut from 3’ to 5’

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Polynucleotide phosphorylases (PNPases)

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break phosphodiester bonds by adding in a second phosphate group, work in 3’ to 5’ direction (an exonuclease)

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Transfer RNA:

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- Decipher the genetic code by linking together an RNA anti-codon (in tRNA) to the RNA codon in the mRNA
- Delivers the appropriate Amino Acid to the proper place in the ribosome when adding to a peptide chain

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Anti-codon:

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specifies which amino acid gets put into what position in the peptide chain. dependent on pairing with mRNA sequence.

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Codon:

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the 5’ to 3’ mRNA strand

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Charged tRNA:

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a tRNA molecule that carries an amino acid to the ribosome to continue protein synthesis. the presence of carrying Amino Acid is what creates a charge on the tRNA molecule.

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Peptidyl transferase:

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Assists Ribosome in forming peptide bonds between amino acids during translation.

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Ribosomal RNA:

Back 38

- Structural component of ribosome which are essential for proper structure
- Participate in the chemical reaction of forming peptide bonds in a growing chain or amino acid

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How are mature/functional tRNA is processed from larger transcripts

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Things to remember:
1. 5’ end has a monophosphate not a triphospate, this increases stability. tRNA and rRNA are more stable then mRNA because they are used over and over again.
2. Mature form is smaller than primary. Use cell lysates to make proteins without using living cells transcript

Steps in Processing:
1. Trim from 3’ end and end up with CCA and 3’ OH
2. Trim from 5’ end, end with GGG and 5’ monophosphate (normal is triphosphate)
3. Modify bases to become unusual- for protection and degradation
4. Add an amino acid to the sugar at the 3’ OH to charge the molecule

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Reporter assay:

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Lets you measure the activity of various enhancer or promoter elements by using luciferase (which takes a substrate, luciferin, and converts it into visible light. **More luciferase means promoter was more active. Less luciferase means promoter was less active

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Transfection:

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Introduction of foreign DNA into eukaryotic cells.

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Transcriptional regulation:

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DNA sequences that are usually upstream (-) of the transcriptional start site bind specific proteins. These proteins mediate expression of the gene. Under different conditions, different proteins will be produced that can bind to those regions. Call these regions regulatory regions: Promoters/Enhancers/Operators (bacteria only)

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cDNA:

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complementary DNA. used to study proteins. * when you make a cDNA you have to bind it to a promoter

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In vitro vs. in vivo

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In vitro: made in a test tube
In vivo: use living cells

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Main differences in eukaryotic vs. prokaryotic gene expression

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1. # of RNA polymerases used: 3 of them in eukaryotes, 1 main polymerase in prokaryotes
2. Complexity of RNA polymerase: more subunits in eukaryotes
3. Prokaryotic RNA polymerase binds to DNA directly; eukaryotic does not
4. Eukaryotes have a nucleus and must export mRNAs to cytoplasm
5. Eukaryotes have nucleosomes and chromatin; prokaryotes do not.
6. mRNA processing is only in eukaryotes.

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Categories of transcripts produced by the 3 eukaryotic polymerases:

Back 46

RNA Polymerase 1: Pre-rRNA (leading to 5.8S, 18S, and 28S rRNA)= Transcribe large rRNAs

RNA Polymerase 2: Pre-mRNA and some snRNAs= Transcribe mRNAs and other non-coding RNAs

RNA Polymerase 3: tRNA, 5S rRNA, U6 snRNA (spliceosome), and 7SL RNA (signal recognition particle).= Transcribe tRNAs, small rRNAS, other non-coding RNAs

Front 47

Core promoter:

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-40 to +40 portion of the template is considered the “core promoter” area for RNA pol II

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S1 nuclease mapping:

Back 48

provides an estimate of the transcription start site

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Initiator sequence:

Back 49

sequence surrounding and including start site of transcription; pyrimidine rich

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TATA box:

Back 50

Similar to the bacterial -10 Binds TBP (TATA-Binding Protein), which is a component of TFIID, the first transcription factor (TF) to bind DNA to initiate transcription sequence . Binds TBP (TATA-Binding Protein), which is a component of TFIID, the first transcription factor (TF) to bind DNA to initiate transcription

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Methods to map the initiation site of transcription:

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S1 nuclease mapping
Runoff transcription
Primer extension

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S1 nuclease mapping:

Back 52

In advance you must know the sequence of the RNA you are looking for
_ There are many RNA molecules present. Synthesize complementary, labeled ssDNA probe of the RNA that you are interested in by reverse transcription.
_ Hybridize your radio active probe to the desired RNAs
_ Digest with S1 nuclease
_ Only the DNA:RNA hybrid molecules (double stranded areas) escape being digested.
_ Run it through a gel and determine the size of the leftover (undigested) DNA.

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Run-off transcription:

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- Clone DNA sequence of interest (including promoter) into a plasmid vector
- Cut DNA at known downstream site to define 3' end.
- Perform in vitro transcription; RNAP falls off at known 3' end.
- Determine size of RNA molecule by gel electrophoresis
- Polyacrylamide gels best of determining exact size.

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Primer extension:

Back 54

- In advance you must know the sequence you are looking for. There are many RNA molecules present.
- Make a gene specific primer for the RNA of interest. You know exactly where it binds.
- Reverse transcribe the cDNA of that RNA
- Run the cDNA on a gel (perform DNA sequencing on the cDNA.)

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Activator:

Back 55

Bind to regulatory promoters and enhancers

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Mediator complex:

Back 56

A large (~30 protein) complex that transmits signals from upstream activating sequences (UAS) to the promoter. Basically, the go-between complex that brings RNAP into position by first recognizing activator proteins in the UAS

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Co-activator:

Back 57

Do not bind DNA directly, but help distal DNA-bound TFs to interact with basal TFs at the promoter

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3 main control regions for transcription

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Core promoters
Regulatory promoters
Enhancers

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Core promoters:

Back 59

Located at direct vicinity of transcriptional start point, where DNA polymerase and transcription factors bind

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Enhancers:

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Far away from the promoter (up or down), can still function when removed and reinserted in opposite orientation, multiple protein-binding sites

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Regulatory promoters:

Back 61

Located further upstream, where transcriptional activators bind

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Main attributes of transcriptional activators

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•Bind to DNA directly
–But not to core promoter, that is just for basal transcription complex
–These bind to regulatory promoters and enhancers (aka, UASs)
•Contain activation domains to assist in recruiting RNA polymerase II
•Recruit RNA polymerase II indirectly through the Mediator complex
•Necessary for efficient transcription (multiple rounds of initiation)

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4 methods to map out DNA sequences involved in transcription initiation

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- DNA affinity chromatography (purify DNA-binding proteins)
- Deletion of various promoter regions
- Linker scanning mutagenesis
- chIP assay

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4 common DNA-binding motifs

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1. Helix-turn-helix
2. Zinc fingers
3. Leucine zippers
4. Helix-loop-helix

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Activator:

Back 65

Bind to regulatory promoters and enhancers

Front 66

Mediator complex:

Back 66

A large (~30 protein) complex that transmits signals from upstream activating sequences (UAS) to the promoter. Basically, the go-between complex that brings RNAP into position by first recognizing activator proteins in the UAS

Front 67

Co-activator:

Back 67

Do not bind DNA directly, but help distal DNA-bound TFs to interact with basal TFs at the promoter

Front 68

3 main control regions for transcription

Back 68

Core promoters
Regulatory promoters
Enhancers

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Core promoters:

Back 69

Located at direct vicinity of transcriptional start point, where DNA polymerase and transcription factors bind

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Enhancers:

Back 70

Far away from the promoter (up or down), can still function when removed and reinserted in opposite orientation, multiple protein-binding sites

Front 71

Regulatory promoters:

Back 71

Located further upstream, where transcriptional activators bind

Front 72

Main attributes of transcriptional activators

Back 72

•Bind to DNA directly
–But not to core promoter, that is just for basal transcription complex
–These bind to regulatory promoters and enhancers (aka, UASs)
•Contain activation domains to assist in recruiting RNA polymerase II
•Recruit RNA polymerase II indirectly through the Mediator complex
•Necessary for efficient transcription (multiple rounds of initiation)

Front 73

4 methods to map out DNA sequences involved in transcription initiation

Back 73

- DNA affinity chromatography (purify DNA-binding proteins)
- Deletion of various promoter regions
- Linker scanning mutagenesis
- chIP assay

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4 common DNA-binding motifs

Back 74

1. Helix-turn-helix
2. Zinc fingers
3. Leucine zippers
4. Helix-loop-helix

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Heterochromatin:

Back 75

More dense DNA, less accessible for RNA transcription, mostly methylated

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Yeast-two hybrid assay:

Back 76

Detects protein-protein interactions

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Fusion protein:

Back 77

You have artificially ligated two unrelated protein domains into a single protein

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Epigenetics:

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Patterns in gene expression controlled by heritable but potentially reversible changes in chromatin structure

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Acetylation:
Methylation:

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Acetylation: loosens
Methylation: tightens

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Euchromatin:

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Less dense DNA, more available, mostly gene sequences, mostly acetylated

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Difference between the 2 main methods used to make DNA more accessible to transcription
(nucleosome modification and chromatin remodeling):

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- Nucleosome modification: acetylation or methylation of basic amino acids in histone tails, removes positive charge and DNA loosens
- Chromatin remodeling: moving histones to new locations, displacing from molecule or translocating to new position on same molecule

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Oligo(dT) column:

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Used to purify MRNA from other RNA (chromatography)

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mRNA processing:

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Capping, poly-A tail, RNA splicing

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Polyadenylation;

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Adding of poly A tails to 3’ end after being cut

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Capping:

Back 85

5 prime cap added to mature MRNA

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Reasons for eukaryote mRNA processing:

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Stability of mRNA
Nuclear export of mRNA to cytoplasm
Promote translation

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Oligo(dT) column:

Back 87

Used to purify MRNA from other RNA (chromatography)

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Which enzymes/proteins are necessary for mRNA processing and nuclear export

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5’ capping (triphosphatase, capping enzyme), poly A tail (poly(A) polymerase), splicing (spliceosome), cap binding complex, poly (A) binding protein

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Describe the structures of the 5’ cap:

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5’ cap: triphosphate, guanine on end, some 2’ methyl groups, 5’ carbon of cap attached to 5’ carbon of first nucleotide

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mRNA processing:

Back 90

Capping, poly-A tail, RNA splicing

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Describe structure of 3' poly(A) tail:

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3’ poly(A) tail: Adenine, adenine, adenine (200-250 A’s)

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Polyadenylation;

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Adding of poly A tails to 3’ end after being cut

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5 types of alternative splicing:

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1 - An exon has a chance of being removed
2 - In an array of exons, any has a possibility of being removed
3 - An intron has a chance of being retained
4 - An exon has a chance of being spliced at its 3’ end
5 - An exon has a chance of being spliced at its 5’ end

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Capping:

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5 prime cap added to mature MRNA

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Reasons for eukaryote mRNA processing:

Back 95

Stability of mRNA
Nuclear export of mRNA to cytoplasm
Promote translation

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Which enzymes/proteins are necessary for mRNA processing and nuclear export

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5’ capping (triphosphatase, capping enzyme), poly A tail (poly(A) polymerase), splicing (spliceosome), cap binding complex, poly (A) binding protein

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Describe the structures of the 5’ cap:

Back 97

5’ cap: triphosphate, guanine on end, some 2’ methyl groups, 5’ carbon of cap attached to 5’ carbon of first nucleotide

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Describe structure of 3' poly(A) tail:

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3’ poly(A) tail: Adenine, adenine, adenine (200-250 A’s)

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5 types of alternative splicing:

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1 - An exon has a chance of being removed
2 - In an array of exons, any has a possibility of being removed
3 - An intron has a chance of being retained
4 - An exon has a chance of being spliced at its 3’ end
5 - An exon has a chance of being spliced at its 5’ end

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Spliceosome:

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Uses a complex of proteins and snRNAs (small nuclear RNA) to find and remove introns. Introns removed by spliceosome are called nuclear introns, because splicing occurs in nucleus. More common

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snRNP:

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small nuclear ribonucleoproteins

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snRNA:

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small nuclear ribonucleic acids

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Branchpoint:

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Found in the middle area of the intron. This is where the 5’ side of the intron after it has formed into lariat structure interacts. A

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Transesterification:

Back 104

The two coordinated transesterification steps in splicing.

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Self-splicing:

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Uses protein complex (no snRNAs) to assist in removing introns. Catalytic activity found within the intron itself. Introns that catalyze their own removal are called group 1 and group 2 introns. splicing in nucleus, less common.

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Process of how introns are removed:

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1. Cut at the 5’ site and form the lariat by 5’-2’ bond connecting the intron 5’G to the 2’ of A at the branch site
2. Cut at the 3’ of the left side exon. This releases the larit strucutre ( intron)
3. Left Exon, 3’ end hybridizes with 5’ of right exon.
4. Intron debranches

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UTR:

Back 107

Untranslated regions at 5’ and 3’ ends before start and after stop codon

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DICER:

Back 108

cleaves RNA down to smaller size

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Guide strand:

Back 109

Short interfering RNA (siRNA): this is the final RNA product after processing miRNA or shRNA. Starts out double-stranded, but then one strand is removed and the remaining strand is the guide strand. Guide strand is complementary and anti-parallel to the target mRNA.

Front 110

siRNA:

Back 110

the functional RNA, (short interfering RNA); let machinary produce most effective siRNA from a larger molecule

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shRNA:

Back 111

this along with miRNA are precursors to make siRNA- which is the functional RNA

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miRNA:

Back 112

micro RNA, longer than shRNA- is the “natural source” wheras shRNA is synthetic source

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RNA interference:

Back 113

a way to regulate gene expression by cleaving specific mRNAs before they are translated or blocking accessibility to ribosomes

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Polyadenylation site:

Back 114

Site that is cut; tail is added to new end
--No consensus sequence here, but ~halfway between the AAUAAA & U-rich sequences

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Polyadenylation signal:

Back 115

Site that is initially recognized; required for cleavage and poly(A) addition

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R2D2:

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Binds to dsRNA, recruites argonaute

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Argonaute:

Back 117

Enzyme that cuts target RNA molecules on guide strand