1
Q
In DNA, what nucleotides bind to each other?
A
A-T
G-C
2
Q
Describe the structure of the DNA backbone.
A
Ribose sugar and Phosphate
Asymmetrical structure
5’ to 3’ from carbons to sugars
Affects replication and transcription
3
Q
Why are DNA strands written 5’ to 3’?
A
Because DNA/RNA are synthesized from the 5’ end
4
Q
What is the central dogma?
A
The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
[DNA]==transcription==>[RNA]==translation==>[amino acid chain]==folding==>[protein]
5
Q
How does RNA differ from DNA?
A
(1) The nucleotides used (U vs T)
(2) the sugar used (ribose vs deoxyribose)
(3) their function (various vs data storage)
(4) their state (single vs double stranded)
6
Q
Compare pyrimidines and purines.
A
Pyrimidine ribonucleotides: Cytosine and Uracil (thymine opposite)
Purine ribonucleotides: Adenosine and Guanosine
7
Q
What are some of RNA molecules’ involvement in the central dogma?
A
Coding:
Messenger RNAs (mRNAs)– carries genetic information from DNA to the ribosomes
Non-coding:
Small nuclear RNAs (snRNAs)– structural components of spliceosomes
Transfer RNAs (tRNAs)– adaptors between amino acids and mRNA codons
Ribosomal RNAs (rRNAs)– structural and catalytic components of ribosomes
Micro RNAs (miRNAs)– short single-stranded RNAs that block expression of complementary mRNAs
8
Q
What is the transcription bubble?
A
Comprised of:
- connection of RNA and DNA template strand
- RNA polymerase
- locally unwound segment of DNA
9
Q
Summarize the process of transcription.
A
(1) DNA is unwound
(2) RNA is synthesized following DNA sequence by RNA polymerase 5’ to 3’
(3) DNA rewinds
(4) mRNA is released
10
Q
On DNA, which strand is coding and which is non-coding?
A
5’ to 3’: nontemplate coding strand
3’ to 5’: template noncoding strand
11
Q
RNA is complementary to the ________ strand, except U in the place of T. RNA is synthesized off the _____________ strand.
RNA is identical to the _______ strand, except U in the place of T.
A
template; non-template; coding
12
Q
True or false? Both DNA strands can serves as a template for RNA. Why?
A
True
The template strand can be different for different genes; both DNA strands of a chromosome serve as the template for transcription
No matter what strand is used, polymerization/synthesis always follows 5’ to 3’
13
Q
Why is RNA produced by transcription of the template strand referred to as sense (+) RNA?
A
Because it contains the correct sequence for translation of mRNA
14
Q
Why can several RNAs be transcribed from the same gene template at the same time?
A
Transcription of housekeeping genes happens non-stop and often involves several template loci in the genome (multiple gene copies).
Genes like ribosomal RNA genes are intensively and continuously transcribed.
Housekeeping genes (required for basic cell function) are always (constitutively) active
For example, actin
15
Q
How does RNA synthesis differ from DNA synthesis?
A
- the precursors are ribonucleoside triphosphate (not deoxy)
- only one strand of DNA is used as a template
- RNA chains can be initiated without a primer
- uracil instead of thymine
16
Q
What is RNA synthesis?
A
The process of synthesizing RNA from the genetic information encoded by DNA is called transcription.
- the RNA molecules will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA coding (sense) strand
- RNA synthesis is catalyzed by RNA polymerases and ALWAYS proceeds in the 5’ to 3’ direction
17
Q
Which direction is RNA formed in?
A
5’ to 3’
18
Q
True or false? Translation copies data from DNA to RNA.
A
False.
Transcription copies data from DNA to RNA
19
Q
Where is RNA made?
A
RNA is synthesized in the nucleus, transported to the cytoplasm
(1) RNA strands start in the nucleus
(2) RNA strands spread to the cytoplasm
20
Q
What is RNA transcribed from?
A
The template strand of DNA (5’ to 3’)
21
Q
How does transcription and translation differ in prokaryotes and eukaryotes?
A
Prokaryotes do not have a nucleus like eukaryotes so the location that the processes take place is different
Prokaryotes: mRNA made from DNA in the nucleiod, chains of amino acids are built (translation) while the transcription process is taking place
Eukaryotes: transcription process takes place within the nucleus and mRNA is transported out into the cytoplasm before translation compenses
22
Q
Summarize the process of gene expression.
A
Enzymatic process 1: Transcription (DNA to RNA)
Enzymatic process 2: Translation (RNA to protein)
23
Q
Gene expression is much simpler in prokaryotes than eukaryotes. Why is this?
A
Eukaryotes:
(1) gene transcription
(2) mRNA processing
- 5’ capping
- Polymerization of poly A tail
- intron splicing
(3) mRNA export
(4) escape from RNAi
(5) mRNA translation
Prokaryotes:
(1) Gene transcription
(2) mRNA translation
This is because prokaryote transcription occurs with translation
24
Q
What are the three stages of transcription in prokaryotes?
A
(1) RNA chain initiation, where RNA polymerase binds to the DNA template strand
(2) RNA chain elongation, where mRNA is built
(3) RNA chain termination
25
What are the functions of the RNA polymerase subunits?
Hint: alpha, beta, omega, and sigma
Sigma (looks like an o): initiation of transcription (released after)
Alpha (looks like an a): assembly of the tetrameric core
beta (looks like a B): ribonucleoside triphosphate binding site
beta prime (B'): DNA template binding region
Omega (looks like a w): chaperone activity controls correct folding of B'
26
What are the stages of Transcription I?
(a) RNA polymerase binds to promoter region
27
What are promoters?
Promoters are composed of an orderly sequence of cis elements (short DNA sequences) that are recognized directly by the RNA polymerase sigma subunit.
Note: promoters are only present on the template strand, which ensures the proper sense mRNA is made
28
Compare cis and trans elements.
Cis-regulatory elements, such as promoters, enhancers, and silencers, are regions of non-coding DNA, which regulate the transcription of nearby genes. In contrast, trans-regulatory factors regulate (or modify) the expression of distant genes by combining with their target sequences
The term cis is derived from the Latin root “cis,” meaning “the same side as.” In contrast, the term trans comes from the Latin root “trans,” meaning “across from.” In molecular biology, a cis-acting (or cis-regulatory) element refers to a region of the chromosomal DNA that regulates the transcription or expression of a gene that is on the same chromosome. A trans-acting (or trans-regulatory) element, on the other hand, refers to a soluble protein that binds to the cis-acting element of a gene to control its expression. The gene that encodes the soluble trans-acting protein can reside on any chromosome, often located far away from the gene whose expression it regulates.
29
Transcription is initiated from a promoter sequence. Explain this in greater detail.
- Promoter sequences vary between genes, with conserved sequence regions
30
Where does transcription begin (after the promoter) and what type of region is this?
Transcription begins at the +1 site, located between the promoter and the ATG start codon. This is a non-coding region.
There are 2 non-coding regions of the mRNA transcript:
5'UTR before AUG and 3'UTR after stop codon
31
Describe the initiation of RNA chains.
(1) RNA polymerase binds directly to promoter region in DNA
(2) RNA polymerase unwinds the two DNA strands to expose a single-stranded template (this costs energy, ATP)
(3) Formation of phosphodiester bonds between the first few rubonucleotides in the nascent RNA chain
32
Describe elongation in transcription.
(1) RNA chain grows from 5' to 3'
(2) RNA polymerase continues to unwind DNA; includes helicase acitivity
(3) DNA re-winding reforming hydrogen bonds between the two DNA strands
33
Describe the final stage of transcription: Termination.
(1) RNA polymerase decouples from DNA template, RNA strand is released
(a) Factor-dependent termination: requires a trans element (p-dependent)
(b) Intrinsic termination: required cis elements in the end of transcription (p-independent)
34
Describe the 4 steps of Rho (p)-dependent termination.
35
Describe intrinsic (p-independent) termination.
End of gene (3') contains a GC-rich inverted repeats section followed by a Poly-A sequence
36
Summarize transciption and RNA processing in Eukaryotes.
Five RNA polymerases in eukaryotes (2 are plant specific)
Post-transcriptional modifications (editing) of mRNAs:
- intron splicing
- 5' capping
- 3' Poly-adenylation
37
What are the key differences between prokaryotic and eukaryotic transcription?
Prokaryotic:
One RNA polymerase
Sigma factors
Co-transcriptional translation
Simple transcript
DNA (almost naked)
Eukaryotic:
Three RNA polymerase
multi-subunit general transcription factors
spatially separated transcription and translation
processed transcript (cap, introns/exons, poly A tail)
chromatin
38
What special challenges make eukaryotic transcription difficult?
(1) Harder to locate promoter: genome is bigger, genes are more spaced out
(2) Transcription and translation are decoupled: different cellular compartments (nucleus and cytoplasm)
(3) Eukaryotic DNA is wrapped up around proteins: histones that need to be removed/moved away for transcription to occur
(4) Eukaryotic transcription is more complex
(a) polymerase has more subunits
(b) transcription factors are required to recruit the polymerase
(I) Polymerase alone cannot find and associate to promoters unlike in prokaryotes with Sigma
(c) there are several types of RNA polymerases
39
Different RNA polymerases specialize in...
subsets of gene templates
All 5 RNA polymerases require transcription factors to initiate transcription
40
Describe the function of RNA polymerase II
Transcribes mRNA and some functional (non-coding) RNAs
Assisted by transcription factors– protein complexes that help it recognize and initiate transcription at the promoter
Most promoters contain a TATA box:
These "TATA"-less promoters use other elements to direct RNA Polymerase II
41
Example of a eukaryotic promoter.
- different conserved regions in promoters depending in the gene
- genes with similar regulation will have similar promoter sequences
- TATA box is important
42
Describe transcription initiation in eukaryotes
(1) Ordered addition of transcription factors forms pre-initiation complex
(2) RNA polymerase starts synthesis and leaves the promoter and TFIID behind
(3) Phosphorylation of RNA polymerase C-terminal domain (CTD) recruits mRNA processing proteins in order
- CTD-domain at -C end of a protein
- capping, splicing, and poly-adenylation
43
What is the process of capping and purpose does it serve?
Co-transcriptional processing of RNA during elongation
Unusual 5'-5' phosphodiester bond of a methylated guanine nucleotide to the first transcribed nucleotide in the RNA
(1) Protects mRNA from nucleases
(2) Recognition signal for translation
44
What is splicing?
Co-transcriptional processing of RNA during elongation:
- Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons
- Introns are excised from the RNA transcripts prior to their transport to the cytoplasm
45
Why are RNA/DNA hybrids more stable than DNA/DNA double stranded molecules?
If you put the DNA of a gene in with mature mRNA of the same gene, RNA will displace dsDNA to make RNA/DNA gene but with weird loops.
The DNA loops are noncoding regions on in the mRNA called introns
they are removed via splicing
46
What are introns and exons?
Only eukaryotes have introns (certain viruses carry sequences from the host eukaryotic genomes with introns).
Introns (or intragenic regions) are noncoding sequences located between coding sequences.
Introns are removed from the pre-mRNA and are not present in the processed (or mature) mRNA.
Exons (expressed regions) are composed of the sequences that remain in the mature mRNA after splicing.
Introns are variable in size and may be very large.
47
Describe the process of splicing (the removal of introns).
The removal of introns must be very precise: should expect clear, conserved sequences.
Actually hard to detect, with minimal conserved sequences.
- Dinucleotide sequences at the 5' and 3' ends of introns (splce sites)
- Branch Point Adenine: for alternative splicing
- TACTAAC box about 30 nucleotides upstream from the 3' splice site (pyrimidine track)
48
What is a homozygote?
When each copy of the gene has the same allele
49
There are two main mechanisms of splicing evolved in eukaryotes. What are they and how do they compare?
(1) Self-splicing: primary transcript with enzymatic activity (ribozyme). No protein involvement so there is no energy required.
(2) RNA/protein complex-mediated splicing:
enzymes/snRNAs needed to recognize and mediate intron excision (spliceosome) so reconfiguration of the splicing machinery requires ATP
50
Give an example of self-splicing and explain.
In some protozoa, introns splice themselves using a guanosine co-factor
51
What is a co-factor?
a compound or chemical used to catalyze a reaction (not a protein)
52
Describe spliceosome-dependent splicing.
snRNA protein structures cut introns from RNA before its transported out of the nucleus
RNA/protein structure excises introns from nuclear pre-mRNA.
There are five snRNAs (U1, U2, U4, U5, and U6) which are small nuclear RNAs.
Some snRNAs associate with proteins to form SNRP (small nuclear ribonucleoproteins)
53
Summarize spliceosome assembly and function of U2 type splicing.
(1) snRNA in the SNRPs bind the transcript through nucleotide interactions with the conserved splicing sites. This facilitates intron removal and brings the exons in close proximity for the reactions to occur.
(2) Complex order of events in each exon/intron/exon boundary that loop the intron segment
(3) Intron sequences are removed with the formation of a loop, called the lariat
54
Which of the following is true about splicing?
(a) Only present in eukaryotes
(b) Can be done without proteins
(c) Is a huge source of disease from mistakes
(d) Occurs during transcription
All of the above
55
60% of disease-causing mutations in humans affect slicing (non-coding sequences). Abnormal splicing is common in cancer cells.
Compare Cis, spliceosome, and trans mutations.
Cis mutations are in the DNA itself
Spliceosome mutations are in the spliceosome itself
Trans mutations are in the genes involved in the proteins doing the splicing
56
Splicing evolved early in eukaryotes– what is it's purpose?
Genes are interrupted by noncoding sequences and introns must be excised perfectly or risk a frameshift error. High risk high reward.
The generation of alternative transcripts via splicing explains how protein diversity can arise from transcription of a limited number of template genes.
The functional relevance of most of these transcripts is not yet known.
More complex organisms have more introns per gene.
57
What is alternative splicing? How does it produce isoforms?
Alternative splicing is when different exons remain after introns are spliced so that a singular DNA sequence codes for two splice variants.
These variants are called isoforms: related but distinct proteins
58
What is poly adenylation?
co-transcriptional processing of RNA during elongation
59
Describe polyadenylation.
RNA polymerase stalls at the end of transcription signal (3' end rich in GT).
Endonuclease activity cleaves transcript downstream of the AU rich region.
Poly A polymerase recognizes processed transcripts as templates to add ~200 adenines without a DNA template (poly A tail)
60
What is the purpose of the poly A tail?
(1) Enhances mRNA stability in the cytoplasm
(2) Mediates mRNA transport across the nuclear envelope
61
What does a mature mRNA, ready to be exported to the cytoplasm, look like?
Cap on 5' end, only coding sequences (introns removed), and 3' poly-A tail
62
Describe the 'life span' of mRNA
mRNA synthesis is rapidly induced and rapidly used up before its degradation in the cell
mRNA are short lived mobile blueprint molecules for protein synthesis
63
Which of the following is not true about translation?
(a) occurs in both eukaryotes and prokaryotes
(b) requires ribosomes
(c) all RNAs are translated
(d) Produces amino acid chains
All RNAs are not translated
64
Is RNA processed in prokaryotes during transcription?
No, only in eukaryotes
65
How does translation differ between eukaryotes and prokaryotes?
Translation occurs at the same time as transcription in prokaryotes because they lack a nucleus.
In eukaryotes, translation occurs after the RNA is transported out of the nucleus into the cytoplasm.
66
What is translation?
The decoding of the code molecule mRNA by tRNA and ribosomes (protein + rRNA)
67
Which of the following are used during translation?
snRNA
tRNA
siRNA
RNAi
none of the above
tRNA
68
What is the likelihood that R is a carrier?
But R is not aa, that is why P= 2/3 and not 1/2
69
Translation involves RNA polymerase, polymerase II, and Polymerase III RNAs. What is the role of each category of RNAs?
70
Describe the structure of transfer RNAs (tRNAs).
- tRNA folds to form specific 3D structures, common among tRNAs
- the 3D structure of the tRNA is important for its function
- serves as substrate for aa linkage
- enters and moves across ribosomal compartments
71
What is an aminoacyl tRNA and how is it formed?
tRNA + amino acid = aminioacyl tRNA
An aminoacyl-tRNA synthetase (ATS) attaches an amino acid to its specific tRNA
There are 21 different ATS's, one for each amino acid that specifically interacts with its corresponding tRNA
72
Explain the specificity of tRNA function.
The specificity of a tRNA depends on matching the correct residue (aa) to the corresponding anticodon.
The aa specificity depends primarily on the activity of aminoacyl tRNA synthases: connects the right amino acid to the right tRNA.
The codon specificity resides on the sequence in the tRNA gene; both must match to allow the genetic code to accurately decode DNA into protein.
73
What two possible outcomes would occur if the residue information and the anticodon information did not match in a tRNA?
(1) The tRNA would not add an amino acid because the aa and codon do not match. The amino acid/3' tRNA plays a role in translation.
(2) The tRNA will introduce the incorrect amino acid even though the codon doesn't match. Only the codon/anticodon pairing is required during translation.
74
Describe the structure of ribosomes
Consist of large and small subunits
rRNAs are structural and have many proteins attached
Svedberg (S) unit of density
E- exit, where the tRNA lands after it has given up its amino acid and where it floats off from
A- starting site or landing pad, tRNA with amino acid attached binds to the A site, this where the anticodon binds correctly with the codon
P- amino acid is going to bind to the growing polypeptide chain found at ths site, the peptide is formed, where the growing protein is actually bound
75
How is the structure of rRNA formed?
folds up by intramolecular base pairing
Note that the ribosome structure and function relies on proper rRNA and tRNA folding
76
What are the three main stages or mechanisms of translation?
(1) Polypeptide Chain Initiation
(2) Chain Elongation (peptide bonds)
(3) Chain termination
77
What is the Shine-Dalgarno sequence in translation initiation in prokaryotes?
In bacteria, the Shine-Dalgarno sequence in the 5' UTR of mRNAs (upstream of the first AUG start codon) binds to the complementary sequence in the 16s rRNA.
- docks the small ribosomal subunit in place
- initial stage of ribosomal assembly needed for translation to start
- with the help of initiation factors (proteins)
78
Describe translation initiation in prokaryotes.
(1) Small subunit (30S) binds Shine-Dalgarno sequence with the help of initiation factors
(2) a special formyl-methionine (fMet) tRNA only used for initiation binds to P site with initiation factors
(3) Large subunit (50S) binds to 30S with the help of initiation factors
79
Describe translation initiation in eukaryotes
(1) Small subunit (40S) binds to Met-tRNA in P-site
(2) Small subunit (40S) binds to mRNA 5'-cap
(3) Small subunit 'walks' along mRNA to start codon (AUG) and lands at P site
(4) Large subunit (60S) binds to 40S
all with the help of initiation factors`
80
What are the binding sites in the elongation stage of translation?
A- aminoacyl binding site
P- peptidyl binding site
E- exit binding site
81
Describe the termination of translation.
(1) Release factor binds A site with stop codon
(2) Translation machinery disassembles
82
What codons are the only codons in the genetic code that do no have a corresponding tRNA?
stop codons
83
A ribosome is mutated so there is no functional A site, what is the first result of that mutation?
elongation of translation cannot occur
84
Decoding mRNA happens...
(a) at the aminoacyl (A) binding site
(b) at the peptidyl (P) binding site
(c) at the exit (E) site
During translation of the ribosome
- A site
85
How many nucleotides to produce an amino acid?
3
86
What establishes the frame of a DNA sequence?
The first ATG (Met) in the mRNA establishes the frame
87
Which of the following errors will be least serious?
1bp deletion
2bp deletion
3bp deletion
a silent mutation
A silent mutation has no effect on the amino acid chain but the 3bp is least serious of the bp deletions
88
How was the codon/aminoacid correspondence revealed?
In vitro translation experiments using synthetic mRNA with a known sequence and labelled amino acids (with radioactive residues)
89
There is degeneracy of the nucleotide in the _______ position of the codon.
third
90
What are six properties of the genetic code?
(1) composed of nucleotide triplets
(2) non-overlapping (coding sequences are never shared between genes)
(3) comme-free (a mature transcript carries the whole, non stop, coding sequence)
(4) is degenerate (there are more than one codon for a given amino acid)
(5) contains start and stop codons
(6) is nearly universal
91
What are the two purines?
Adenine and Guanine
92
What are the two pyrimidines?
Cytosine and Uracil
93
What are the wobble rules of the third base Codon-anticodon pairings?
The wobble rules identifies base pair interactions between mRNA (3' end of codon) and tRNA (5' end of codon) that do not follow normal pairing rules (A-t and C-G).
Hypoxanthine (I) is a purine derivative formed by deamination of adenine. It is found only in anticodons of some tRNAs where it can associate with U, C and A in the codon of mRNAs.
Some tRNAs can interact with more than one codon, this is because position one and two are essential but the third is not that important. Degeneracy of the nucleotide in the third position of the codon.
94
What effect do mutations that introduce a premature stop codon in the transcript cause?
truncated (short) version of the protein
this often leads to lack of function and a mutant phenotype
95
What are suppression mutations?
A mutation that 'fixes' another mutation without directly reversing it.
A cell has a premature stop codon producing truncated proteins but a tRNA mutation ignores that stop codon allowing the full transcript. The novel tRNA gene changes the anticodon region such that it can now recognize the STOP codon as an amino acid site, bypassing early termination.
96
A ________ counteracts the effects of a nonsense mutation,
suppressor
97
Leu, Val
98
How does RNA replicate itself?
RNA genetic material is used as a template to generate sense mRNA for protein synthesis but also as a template to replicate its genome material.
Both are accomplished without DNA via the activity of RdRP enzymes.
99
How is RNA retrotranscribed to DNA through reversetranscriptase activity in retroviruses.
RNA viruses that use sense RNA as their genetic material produce a DNA copy that integrates in the host cell's genome using reverse transcriptase that polymerizes DNA from RNA templates.
100
What is a prion?
Protein + infection = prion
Self-reproducing pathogenic proteins
101
What is the catalyst in the chain reaction of PrPc to PrPsc conversion?
PrPsc as a catalyst
102
Summarize the central dogma after the knowledge of topic 5.
BIOL226
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