What are three major differences between an mRNA and a pre-mRNA?
1. pre-mRNA is not spliced while mRNA is
2. pre-mRNA lacks a poly-a tail
3. mRNA carries genetic code
What are the basic steps of transcription, briefly describe what happens at each step.
1. Recognition- recognition of the core promoters/TATA Box TF11D binds at promoter and recruits other transcription factors for recognition of promoter by RNA Pol.
2. Initiation- formation of the preinitiation complex, binding of RNA Pol.
3. Elongation- m
For a eukaryotic protein coding gene, list the steps of mRNA processing and briefly describe what happens at each step.
addition of 5' cap consists of the addition of an extra nucleotide at the 5' end of the mRNA and methylation by the addition of a methyl group (CH3) to the base in the newly added nucleotide and to the 2'-OH of the sugar of one or more nucleotides at the
Explain verbally or with a diagram how specific transcription factors that bind enhancers far away from the core promoter can up regulate transcription. Include necessary protein complexes in your explanation/drawing.
Activator proteins bind to the enhancer and cause the DNA between and the promoter to loop out, bringing the promoter and enhancer close to each other, and so the transcriptional activator proteins are able to directly interact with the basal transcriptio
Two steps of RNA processing are coordinated with transcription, what are they, which steps of transcription are the coordinated with, and what part of RNA Pol II is required for the first of these steps to occur?
1. 5' cap: functions in initiation of transcription
2. 3' poly A tail: export, translation, and stability (protection from degradation); coordinated with termination
What happens to the lariat after the two exons are ligated together?
The lariat gets degraded. The intron gets released as the lariat.
Changes in how telomerase is expressed can result in unconstrained cellular division, contributing to cancer. Approximately 90% of human cancers express telomerase, whereas somatic cells generally do not. There is ongoing research into how inactivating te
A) DNA polymerase cannot replicate and repair DNA molecules at the ends of linear chromosome.
The ends of linear chromosomes, called telomeres, protect genes from getting deleted as cells continue to divide.
The telomerase enzyme attaches to the end of th
In your own words, list a comprehensive definition for "gene" at the molecular level.
A gene is a series of DNA that is translated to RNA, which is expressed as a protein.
At the molecular level: any DNA sequence that is transcribed into an RNA molecule.
Outline in words and diagrams how telomeres at the end of eukaryotic chromosomes are replicated.
Telomerase recognizes an existing telomere and elongates the strand using and RNA template, then DNA Pol Alpha fills in the lagging strand.
Name 3 types of RNA molecules and explain the function of each.
1. rRNA- makes up the ribosome, the site of protein assembly; directs the translation of mRNA into proteins
2. mRNA- mRNA transcibes the genetic code from DNA into a form that can be read and used to make proteins. mRNA carries genetic information from th
Name two unusual phosphodiester bonds that form during mRNA processing.
1. 2'-5' phosphodiester forms the Larient when the introns are spliced.
2. 5'-5' phosphodiester when 5' 7 methyl guanosine cap is added to mRNA
What is "wobble" and what property of the genetic code is it related to? Explain why wobble results in this property.
The wobble is flexible in base pairing at the 3rd position of the codon. There are 30-50 tRNAs = more codons than anticodons
A geneticist discovers that two different proteins are incoded by the same gene. One protein has 56 AAs and the other has 82 AAs. Provide a possible explanation for how the same gene can encode both of these proteins (include a diagram).
Alternative splicing of the pre-mRNA
What three kinds of of damage can different types of radiation cause in DNA? Choose one of these types of damage and explain why it can result in mutation and what types of mutations are expected to occur.
UV Radiation- Purine and pyrimidine bases absorb UV light which leads to the formation of bonds between adjacent pyrimidine molecules on the same strand. These bonds are called pyrimidine dimers. This distorts the configuration of DNA and can block replic
You are conducting an experiment that requires you to use a chemical called "Carcinox." You expect it may cause cancer. Describe how to carry out the Ames test to see if this is true. Be sure to include bacterial genotype, type of media, and results you w
Get some strains of bacteria that are his- and mix it with liver enzymes. Half of it goes onto a petri dish with a medium that lacks histidine. The other half gets mixed with "Carcinox" and also placed on an identical but separate dish. Both dishes are in
What is snRNP ("SNURP") and what role does it play in the cell?
snRNPs- small nucleur ribonucleoproteins. Have an snRNP component (U #) and a protein component. Components of the spliceosome. Helps with processing of RNA (ch10 pg 245).
A yeast strain was exposed to a chemical mutagen. As expected, exposure to mutagen resulted in a DNA sequence change within the coding region of an essential gene you examined. Yet, this mutagen did not result in any lethal phenotype. How could you explai
Mutation in essential gene- non lethal; third base pair wobble probability didn't change the amino acid; silent mutation; could be a neutral mutation- alters a.a. but replaced with one of similar/function so doesn't alter protein function or overall shape
What would be the consequences of a two nucleotide deletion mutation in the middle of the coding region of a protein-coding gene?
Frameshift mutation- could lead to a nonsense codon and early termination; or a completely different gene function, loss of function, or partial loss of function.
Genes can have a default state of "OFF." Explain how this can be true in simple prokaryotic pathway where the only regulatory protein is a repressor.
The activator is inactive. It's a positive control in an inducible operon. pg 295
Why is gene regulation important for bacterial cells?
Because prokaryotic mRNA is not processed before translation. Translation of bacterial cells can begin before transcription is complete, therefore transcription is the key step at which prokaryotes regulate gene expression.
Gene regulation allows for bioc
Why is gene regulation important during cell differentiation?
Alternative splicing allows different mRNA molecules to be produced from pre-mRNA, which in turn yields different proteins. Different proteins lays the groundwork for different cell types and tissue differentiation. For example, in the fruit fly, the tra
Given a diagram of a simple case of constitutive, inducible, or repressible regulation, identify if a prokaryotic gene will be turned on or off.
inducible and repressible are on slides 17-20 on lecture 32.
How are regulatory transcription factors and regulatory splicing factors similar in their mechanism of action?
regulatory transcription factors must bind to the regulatory promoter upstream of the start site in order for transcription to commence. Additionally, they must bind in the correct order. Similarly, regulatory splicing factors (eg. snRNPs) must bind in th
What is the difference between general and specific transcription factors?
General Transcription Factors: required for transcription, all genes (slide 31 lecture 23)
Specific Transcription Factors: stabilize the holoenzyme, sometimes chromatin modification, specific for different genes (regulatory)
Mutations can occur in regulatory motifs that make up the core promoter/enhancers/silencers/insulators. How might such mutations impact gene expression?
...
Arrange the following components of translation in approximate order in which they would appear or be used in protein synthesis: 70S initiation complex, release factors, peptidyl transferase activity, 30S initiation complex, and fMet-tRNA.
fMet-Trnafmet, 30S initiation Complex, 70S initiation complex, peptidyl transferase activity, release factors (Chp. 11 Pg. 279-281)
Name the steps of translation that require energy (in the form of GTP).
1. Initiation
2. Elongation
3.Termination
Explain how initiation of translation differs between prokaryotes and eukaryotes.
In bacterial cells, sequences in 16S rRNA of the small subunit of the ribosome bind to the Shine Dalgarno sequence in mRNA. No analogous consensus sequence exists in eukaryotic mRNA. Instead, the cap at the 5' end of eukaryotic mRNA plays a critical role
Explain the function of the A, P, and E sites during translation.
A sites: the ribosomal site most frequently occupied by the aminoacyl-tRNA.The aminoacyl-tRNA in the A-site functions as the receptor for the growing protein during peptide bond formation.
P-sites: the ribosomal site most frequently occupied by peptidyl-t
The basic structure of an amino acid. Label or draw a cartoon of the components.
...
The genetic code: how many possible codons, how many standard AAs, how many bases in a codon, etc.
Possible codons: 64
Standard AAs: 20
Bases in a Codon: 3
Nonpolar
Type of R Group
Hydrophobic- repels water
alanine (ala-A), valine (val-V), leucine (leu-L), isoleucine (ile-I), proline (pro-P), methionine (met-M), phenylalanine (phe-F), tryptophan (trp-W).
Polar
Type of R Group
Hydrophilic- attracts water
glycine (gly-G), serine (ser-S),threonine (thr-T), cysteine (cys-C), tyrosine (tyr-Y), asparagine (asn-N), glutamine (gln-Q)
Positive Charged
Type of R Group
+
lysine (lys-K), arginine (arg-R), histidine (his-H)
Negative Charged
Type of R Group
-
aspartic acid (asp-D), glutamic acid (glu-E)
What happens when the mutations are somatic vs when they occur in the germ-line?
Somatic- mutations arise in somatic tissues, the mutations are passed to the daughter cells. The earlier in the development the mutation occurs the larger the clone of cells with the mutations gets.
Germ-line Mutations- arise in cells that produce gametes
Base Substitution
Changes the base of a single DNA nucleotide
Transition
Base substitution in which a purine replaces a purine or a pyrimidine replaces a pyrimidine
Transversion
Base substitution in which a purine replaces a pyrimidine or a pyrimidine replaces a purine
Insertion
Addition of one or more nucleotides
Deletion
Deletion or one or more nucleotides
Frameshift Mutation
Insertion or deletion that alters the reading frame of a gene
In-frame deletion or insertion
Deletion or insertion of a multiple of 3 nucleotides that does not alter the reading frame
Expanding Trinucleotide Repeats
Repeated sequence of three nucleotides (trinucleotide) in which the number of copies of the trinucleotide increases
Forward Mutation
Changes the wild-type phenotype to a mutant phenotype
Reverse Mutation
Changes a mutant phenotype back to the wild-type phenotype
Missense Mutation
Changes a sense codon into a different sense codon, resulting in the incorporation of a different amino acid in the protein.
Nonsense Mutation
Changes a sense codon into a nonsense codon, causing premature termination of translation.
Silent Mutation
Changes a sense codon into the synonymous codon, leaving the amino acid sequence of the protein unchanged.
Neutral Mutation
Changes the amino acid sequence of a protein without altering its ability to function.
Loss-of-function Mutation
Causes a complete or partial loss of function
Gain-of-function Mutation
Causes the appearance of a new trait or function or causes the appearance of a trait in inappropriate tissue or at an inappropriate time
Lethal Mutation
Causes premature death
Suppressor Mutation
Suppresses the effect of an earlier mutation at a different site
Intragenic Suppressor Mutation
Suppresses the effect of an earlier mutation within the same gene
Intergenic Suppressor Mutation
Suppresses the effect of an earlier mutation in another gene
Ionizing Radiation
X-rays, gamma rays, and cosmic rays
Capable of penetrating tissues and damaging DNA. They dislodge electrons from atoms they encounter, changing stable molecules into free radicals and reactive ions, which then alter the structures of bases and break phos
Basic Rules of Transcription
Transcription is a selective process
Only certain parts of DNA are transcribed
RNA is transcribed from ssDNA
Within a "gene" only one strand is normally transcribed
RNA messages are antiparallel and are complementary to the DNA template strand
Transcripti
RNA Pol II Core Enzyme
Protein involved in transcription
a multisubunit enzyme (of multiprotein complexes) that catalyzes the synthesis of RNA from a ssDNA template. The core enzyme contains subunits necessary for polymerization of RNA molecules
RNA Pol II Holoenzyme
Protein involved in Transcription
Transcription requires RNA Pol II AND the general transcription factors (for
example TFIIB, TFIIE, TFIIF) + more. The holoenzyme contains all subunits necessary to begin transcription, includes the CTD tail, general trans
Splicing
A) Draw the structure of a typical eukaryotic pre-mRNA molecule transcribed from a gene containing one intron and two exons. Indicate the introns and exon, splice sites, branch point, poly (A) site, 5? UTR, and 3? UTR.
B) Diagram the first reacti
A) Unable to upload picture, see google doc
B) [Forming the loop] The pre-mRNA is cut at the 5' splice site. This cut frees exon 1 from the intron; and the 5' end of the intron attached to the branch point, forming a lariat. The result is now that the 5'
Primary Structure of a Protein
the linear arrangement of amino acids in a protein and the location of covalent linkages such as disulfide bonds between amino acids.The primary structure of a protein can readily be deduced from the nucleotide sequence of the corresponding messenger RNA.
Secondary Structure of a Protein
areas of folding or coiling within a protein; examples include alpha helices and pleated sheets, which are stabilized by hydrogen bonding.
Tertiary Structure of a Protein
the final three-dimensional structure of a protein, which results from a large number of non-covalent interactions between amino acids.
Quaternary Structure of a Protein
non-covalent interactions that bind multiple polypeptides into a single, larger protein. Hemoglobin has quaternary structure due to association of two alpha globin and two beta globin polyproteins.
Alpha Helix
a common secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding).
Beta Sheet
the second form of regular secondary structure in proteins. It is less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A bet
mRNA Processing
A) For a eukaryotic protein coding gene, list the steps of mRNA processing and briefly describe what happens at each step.
B) Two of these processing steps produce modifications that are important for translation. Which are they and what i
A) addition of 5' cap consists of the addition of an extra nucleotide at the 5' end of the mRNA and methylation by the addition of a methyl group (CH3) to the base in the newly added nucleotide and to the 2'-OH of the sugar of one or more nucleotides at t
General Transcription Factors
Proteins involved in transcription
Required for transcription, all genes
TBP
Proteins involved in transcription
TATA Binding Protein- essential component of TFIID
TFIID
Proteins involved in transcription
Binds at the promoter and recruits other general transcription factors that are required for recognition of the promoter by
RNA pol II
TFIIB
Proteins involved in transcription
binds to the BRE element. Involved in initiation, stabilizes the TFIID complex and is involved in recruiting RNA pol II (complexed with other general transcription factors) to the core promoter.
Mediator
Proteins involved in transcription
part of the holoenzyme that interacts with activators
Specific Activators/Transcription Factors
Proteins involved in transcription
stabilize the holoenzyme, sometimes chromatin modification,
specific for different genes (regulatory)
Elongation factors
Proteins involved in transcription
required for processivity. Evict histones ahead of RNAP II and replace them behind it. Maintain heteroduplex RNA:DNA hybrid region. Deal with lesions and problematic sequence (resulting in
bends or if there is complement
Termination Factors
Proteins involved in transcription
Involved in cleaving the RNA and release of RNAPII from the template
Steps of Transcription
0. Open chromatin conformation such that the core promoter can be accessed by general TFs
1. Recognition: of the core promoter, binding of TBP to the TATA box (or nonspecifically if the gene does not contain a TATA box), TFIID complex is recruited (binds
DNA Transporons
Transposable elements that transpose as DNA. Replication requires transposase
Retrotransporons
Elements that transpose through an RNA intermediate. RNA is transcribed from the transposable element (DNA) and is then copied back into DNA by the enzyme "reverse transcriptase
Deletion
Pairing by looping and crossing over between two transposable elements oriented in the same direction
Inversion
Pairing by bending and crossing over between two transposable elements oriented in opposite direction
Misalignment and unequal exchange between transposable elements located on sister chromatids leads to one chromosome with a _____ and one chromosome with a _____.
deletion
duplication
In yeast, transcription of enzymes required for the biochemical pathway converting galactose to glucose-1-phosphate is regulated by GAL4 protein. GAL4 protein promotes transcription by binding to a regulatory motif (called the upstream activator sequence,
A) Most of the enzymes are constitutively expressed. The GAL80 enzyme is inducible.
B) If GAL80 was completely non-functional then there would be no repression of GAL genes.They would be transcripted without regulation
C) since the GAL4 would be upstream
Aminoacyl Synthetases
enzyme that attaches an amino acid to a tRNA in translation. Each aminoacyl-tRNA synthetase is specific for a particular amino acid.
Small Ribosomal Subunit
The smaller of the two units of a functional ribosome.
Large Ribosomal Subunit
The larger of the two units of a functional ribosome
Initiation Factors (as a group)
IF-1: protein required for the initiation of translation in bacterial cells; enhances the disassociation of the large and small subunits of the ribosome
IF-2: protein required for the initiation of translation in bacterial cells; forms a complex with GTP
Elongation Factors (as a group)
EF-Ts: protein that regenerates elongation factor Tu in the elongation stage of protein synthesis
EF-Tu: protein taking part in the elongation stage of protein synthesis; forms a complex with GTP and a charged amino acid and then delivers the charged tRNA
Peptidyl Transferase Activity (ribozyme)
...
Termination Factors (as a group)
...
Capping Enzyme
Proteins involved in RNA processing
the guanylyl transferase that adds guanosine to the 5' end of the pre-mRNA transcript
RNA methyltransferase
Proteins involved in RNA processing
methylates the guanosine at position 7 and several downstream nucleotides
PAP
Proteins involved in RNA processing
Polyadenylate polymerase the enzyme that adds the adenine nucleotides
PABII
Proteins involved in RNA processing
Poly-A binding protein- promotes export from nucleus, translation and stability
CPSF
Proteins involved in RNA processing
Cleavage & polyadenylation specific factor (recognizes AAUAAA)
CSF
Proteins involved in RNA processing
Cleavage stimulation factor (recognizes G/U Rich Region
The Spliceosome
Proteins involved in RNA processing
A large multi-component complex (with changing configuration and membership) that catalyzes the reactions necessary for intron removal
snRNPs
small nuclear ribonucleoporteins. Have an snRNA component (U #) and a protein component. Components of the spliceosome
Ribozymes
Catalytic RNA molecules can cut out parts of their own sequences, connect some RNA molecules together, replicate others, and even catalyze the formation of peptide bonds between amino acids.
pre-MRNAs
the immediate products of transcription in eukaryotic cells. Also called primary transcripts, they are modified extensively before becoming mRNA and exiting the nucleus for translation into protein. Bacterial cells do not possess pre-mRNA; in these cells,
Ribosomal RNA
rRNA
Cell type: eukaryotic and bacterial
location: cytoplasm
Function: structural and functional components of the ribosome
Messenger RNA
mRNA
Cell type: eukaryotic and bacterial
location: nucleus and cytoplasm
Function: carries genetic code for proteins
Transfer RNA
tRNA
Cell type: eukaryotic and bacterial
location: cytoplasm
Function: helps incorporate amino acids into polypeptide chain
Small nuclear RNA
snRNA
Cell type: eukaryotic
location: nucleus
Function: processing if pre-mRNA. combine with small protein subunits to form small nuclear ribonucleoproteins (snRNPs)
Small nucleolar RNA
snoRNA
Cell type: eukaryotic
location: nucleus
Function: processing and assembly of rRNA
MiroRNA
miRNA
Cell type: eukaryotic
location: cytoplasm
Function: inhibits translation of mRNA
Small interfering RNA
siRNA
Cell type: eukaryotic
location: cytoplasm
Function: triggers degradation of the other RNA molecules
Telomerase
binds to DNA at unreplicated 3'-ends and adds base pairs
What would happen if there was no functional telomerase?
Chromosomes would get shorter
The molecular definition of a gene
all the DNA sequence necessary to synthesize a functional RNA molecule
Exons
coding = phenotype
different genes = different exons
Introns
intervening sequences"
areas of genes that do not generally code for a phenotype
the start and end sequences are highly conserved
TATA Box
a highly conserved sequence which serves as the binding site for RNA pol (start site of transcription); key signature of the promoter region of a gene
Regulatory Promoter
Sequence which binds transcriptional activator proteins. Stimulate and stabilize activity of the core promoter. Generally different for different genes.
Regulatory Sequence
specific DNA sequences at which DNA binding proteins (e.g., specific transcription factors) attach and modulate the RATE and COPIES of a gene made - when, where and how much
Enhancers
Regulatory Sequence
enhance transcription rate
Silencers
Regulatory Sequence
reduce transcription rates (Sometimes same site)
Insulators
Regulatory Sequence
boundary sequences that block or 'insulate' promoters from the effects of enhancers or silencers
What is Transcription?
Where: in the nucleus
When: either G0 (no-dividing), G1 or G2 (period when genes coding for cellular organelle proteins are synthesized)
How: Gene Promoter Region (TATA box + more) defines which Nucleotides will be transcribed
It is a selective process wi
A DNA Non-Template strand has the following sequence, 3' -CCAGTTTA- 5'. The pre-mRNA sequence will be...?
GGUCAAAU
Core Promoter
a highly conserved sequence which serves as thebinding site for RNA Pol II (start site of transcription).
TATA box, one of the key signatures of the CORE PROMOTER region of a gene
Regulatory Promoter
Sequence which binds transcriptional activator proteins (Transcription Factors). These stimulate and stabilize activity of the core promoter. These regions are generally different for different genes.
Sequence Positions
Distal Region: -200 bp and upstream
Proximal Region: -50 to -200 bp
Core Region: +40 to -50 bp
No nucleotide 0
+1 is the first nucleotide transcribed
+30 is the thirtieth
-25 is 25 bases before the +1 site
Consensus Sequences
N- any nucleotide
R- A or G (purine)
S- C or G
V- A or C or T
W- A or T
Y- C or T (pyrimidine)
Steps of Transcription
1. Recognition: Promoters
2. Initiation: Binding of RNA Pol
3. Elongation: Movement of RNA Pol
4. Termination: GC rich regions - Poly-A region
Result is all nucleotides of Transcriptional Unit converted from DNA to RNA
RNA Polymerase II
transcribes pre-mRNA, some snRNAs, snoRNA, some miRNAs
multisubunit enzyme (of multiprotein complexes) that catalyzes the synthesis of RNA from a ssDNA template. No primer required, starts at +1.
Shine-Dalgarno Sequence
serves as the ribosome binding site during translation--only in prokaryotes
5' untranslated region (5' UTR)
a sequence of nucleotides at the 5' end of the mRNA that does not encode any of the amino acids of a protein
3' untranslated region (3' UTR)
affects the stability of mRNA and the translation of the mRNA protein-coding sequence
Describe three examples of types of genes that are not protein encoding
Genes include DNA sequences that encode proteins, as well as rRNA, tRNA, snRNA, as well as other types of RNA." I believe it is saying that rRNA, tRNA, and snRNA are three types that don't code for proteins and is therefore what she is asking for. In a r
Genes can have a default state of 'ON'. Explain how this can be true in a simple prokaryotic pathway where the only regulatory protein is a repressor.
Negative control in a repressible operon with an inactive repressor. pg 294
Spontaneous Mutations
mutations that occur under normal conditions
Repeat Expression
section of DNA has lots of repeats. The strands separate and replicate. While being replicated a hairpin loop forms on the strand being synthesized. Part of the template strand gets replicated twice because of this
Non-Homologous Crossover
leading to a Robertsonian translocation
Mispairings due to wobble
Flexibility in the DNA structure is to blame
Strand Slippage
when one nucleotide strand forms a small loop. If the looped out nucleotides are on the newly synthesized strand, an insertion results. The insertion will be replicated durings the next round of replication and then both strands will have the insertion. I
Unequal crossing over
misaligned pairing can cause this. The results are one DNA molecule with an insertion and the other molecule has a deletion
Depurination
the loss of a purine base from a nucleotide. The covalent bond connecting the purine to the 1' - carbon atom of the deoxyribose sugar breaks which produces an apurinic site (a nucleotide without its purine base). This site cannot act as a template for a c
Deamination
the loss of an amino group (NH2) from a base. It can be both induced or spontaneous. Deamination alters pairing properties
Induced mutations
mutations that result from changes caused by environmental chemicals or radiation
Base Analogs
chemicals with structures similar to that of any of the four standard bases of DNA. DNA polymerase can't tell the difference between these
Alkylating Agents
donate methyl and ethyl to nucleotide bases
Hydroxylamine
adds a hydroxyl group to cytosine. Increases the frequency of a rare tautomer. Hydroxylamine can't reverse the mutations it creates
Intercalating Agents
Proflavin, acridine orange, ethidium bromide, and dioxin. They sandwich themselves between adjacent bases in DNA distorting the three-dimensional structure of the helix which causes single-nucleotide insertions and deletions which frequently produce frame