LECTURE 5: mRNA and Transcription

Question 1

List the steps in which genetic information flows within a cell

Answer 1

DNA-> RNA-> Proteins

1. Transcription Copies A Segment Of
DNA (gene) into pre-mRNA
2. The Pre-mRNA is edited into
mature mRNA
3. The mRNA is exported from the
nucleus to the cytoplasm
4. The mRNA is translated by
ribosomes (made of rRNA and proteins) using tRNAs

Question 2

List the three major classes of RNA in a cell

Answer 2

messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA)

Question 3

Gene

Answer 3

A unit of heredity. Usually a segment of DNA that contains the information (nucleotide code) to produce an RNA molecule

Question 4

rRNA

Answer 4

provide structural support to make a ribosome, and catalyzes the chemical reaction in which amino acids are linked to one another.

Question 5

tRNAs

Answer 5

are required to translate information in the mRNA code into amino acids.

Question 6

Describe how the coding works as you go from a DNA sequence to a protein amino acid sequence

Answer 6

Transcription: One of the DNA strands of a gene is used as a template strand to produce an RNA strand. The code for the RNA strand is the complement of the code of the DNA strand. Formed by base-pairing during RNA synthesis.

Translation: The mRNA is read out in triplet basepairs (called a codon) by the ribosome. Each codon codes for a different amino acid. Different tRNAs bind to different codons by using their anticodon, and each tRNA carries a specific amino acid.
e.g. met-tRNA will recognize the codon AUG on the mRNA strand, using its anticodon UAC, and will always carry methionine (M)

Question 7

List which strands are used for each step, and the direction in which the are oriented

Answer 7

DNA: 3’ TO 5’

mRNA: 5’ TO 3’

PROTEINS; 5’ TO 3’

Question 8

List the basic properties of an mRNA molecule

Answer 8

• They each code for a specific polypeptide.
• They are found in the cytoplasm.
• They are attached to ribosomes when translated. Only the coding region is translated into a polypeptide.
• Most have a non-coding segments: (5’ untranslated region and 3’ untranslated region).
• Eukaryotic mRNAs modifications at their 5’ (guanosine cap) and a 3’ poly(A) tail.

Question 9

The coding region consists_______________________________

Answer 9

of an open reading frame (ORF), which is a continuous stretch of codons that code for the polypeptide primary structure.

Question 10

Describe the functions of RNA polymerase II function

Answer 10

i. Unwinds the DNA strands
ii. Reads the DNA template strand in a 3’
to 5’ direction
iii. Catalyzes The Polymerization Of
ribonucleotides in a 5’ to 3’ direction (adds the next base 5’ phosphate to the 3’ hydroxyl of the previous base)
iv. Uses ATP,GTP,CTP,and UTP as substrate

Question 11

List the RNA polymerases that produce larger rRNAs, mRNAs, snRNAs and snoRNAs, and tRNAs

Question 12

RNA polymerases

Answer 12

These are multi-unit enzymes that incorporate nucleotides into a strand of RNA from a DNA template.

Question 13

Which enzyme transcribes the DNA?

Answer 13

RNA Poly 2

Question 14

Compare the process of transcription between Eukaryotes and Prokaryotes

Answer 14

Eubacteria and archaea only have a single RNA pol.The archaea unit more closely resembles eukaryotic RNA pol II

Question 15

Describe the initiation of transcription

Answer 15

The core promoter is just ‘upstream’ of the transcriptional start site and is where RNApol II binds prior to initiating transcription.

RNApol II requires the help of general transcription factors (GTFs) to recognize the part of the promoter and form a pre-initiation complex (PIC)

A critical portion of the eukaryotic promoter lies 24-32 bases upstream from the initiation site, and contains the TATA box.

Question 16

Describe how the pre-initiation complex forms at a general promoter

Answer 16

The preinitiation complex assembly start swith the binding of the TATA-binding protein (TBP) to the promoter.
• TBP is a subunit of the TFIID and when it bind s to the promoter causes a conformation change in DNA.
• Binding of TFIID sets the stage for the assembly of the complete PIC.
• The threeGTFsboundtothepromoterallows the binding of RNA polymerase II with its TFIIF.
• ThespacingoftheTATAboxandtheotherGTS means that RNApolII is positioned right at the transcriptional start site
• AslongasTFIIDremainsboundtothepromoter, additional RNA polymerases may be able to attach for additional rounds of transcription. So multiple mRNAs can be made from a gene at once… amplification. From each of these mRNAs, multiple proteins can be transcribed!

Question 17

List what functions occurs at the C-terminal domain of RNA pol II

Answer 17

The C-terminal domain of the RNAPII serves as a scaffold for other factors involved in RNA processing.
Some of the factors are important in:
•Capping, poly A tailing and intron removal (i.e. splicing).

Question 18

Is this statement T or F:The growing pre-mRNA is processed as it’s
being made

Answer 18

T

Question 19

kinase

Answer 19

is an enzyme that adds a phosphate group to another molecule (often another protein). Addition of the phosphate can alter the other protein’s function!

Question 20

describe the structure and function of the 5’ cap

Answer 20

Cap structure:
• “inverted” guanine
nucleotide added (5’ to 5’
bond)
• Methyl groups added to first
(G) and second nucleotides
• A protein cap is added

Functions of the cap:
• stabilizes the 5’ end,
protecting it from
exonucleases(chew up phosphodiester bonds) 
• aids in transport out of the
nucleus
• Helps to start the translation
process

Question 21

Describe the polyA tail function

Answer 21

onfers stability to the mRNA
• In cytoplasm, polyA tail is gradually removed by polyA ribonuclease
• When <30A s are left, the mRNA is degraded by exonuclease – cuts inward from both ends
• Variable mRNA lifespan (in cytoplasm) influences amount of protein made – translational level control.
• Cellcycleproteins,cyclins,Fos–transcriptslast10-30min
• Major proteins, hemoglobin, ovalbumin - transcripts last 24 hrs.
• Half-life also influenced by specific sequences in 3’UTR • CCUCCrepeatsstabilizemessage.(hemoglobinmRNA)
• AUUUArepeatsde-stabilize(alterationofthisinFOSmRNAcanleadto cancer)… FOS is a transcription factor involved in cell cycle control

Question 22

Describe how polyA tails can be used to isolate mRNA molecules from other molecules

Answer 22

Not all RNAs have poly(A) tails. e.g. rRNA, tRNA, histone encoding genes don’t have them. mRNAs encoding most proteins do (in eukaryotes).

If we’re sequencing a transcriptome, we usually don’t want the sequence of rRNAs etc., only the mRNA sequences.

So, we use magnetic beads with oligo d(T)s attached to them. The d(T)s hybridize with the poly(A) tails of the mRNAs.
You then just use a magnet to pull out the beads and wash away all the rRNA and tRNA, and use the purified poly(A) mRNA.
So this will capture all of the mRNAs that are present in a cell or tissue for gene sequencing

Question 23

Describe how the polyA tail is formed

Answer 23

5’ CAP already added, RNApolII is merrily transcribing the DNA…

Polyadenylation signal = DNA sequence in the 3’ UTR that codes for AAUAAA in the RNA An endonuclease recognizes this sequence and cuts the RNA strand 15bp down from it

Then poly(A) polymerase adds the poly(A) tail to the 3’ end of the RNA molecule. Remember that these enzymes are organized by the RNApol CTD!

any more RNA made by RNApolII gets chopped up because the cap is gone.
- the polymerase eventually stops because it loses general transcription factors

Question 24

List the steps of RTPCR

a. Describe what happens at each step
b. Describe the purpose of RTPCR

Answer 24

Polymerase chain reaction (PCR) is a method to amplify a section of DNA into a bazillion copies that you can do stuff with. It doesn’t work when starting with an RNA molecule (DNA polymerases need a DNA template to work with)

So we first ‘reverse transcribe’ (RT) mRNAs using a reverse transcriptase and an oligo d(T) primer (short piece of DNA)
This produces single-stranded ‘complimentary DNA’ molecules that are, complementary to the RNA molecules.

These can be used as an input into PCR to amplify a specific gene (or section of a gene) you’re interested in. So the whole thing together is ‘RT-PCR’

Question 25

Describe the structures that facilitate intron splicing (both snURPS and consensus sequences splice- sites

Answer 25

Consensus sequence: same for almost all genes small nuclear RNAs (snRNAs) bind to proteins (the U proteins) to form snRNPs - pronounced “snurps” highly conserved, recognizable sequence regions called the splice- sites

Question 26

spliceosome

Answer 26

the complex of snRNA, hnRNA and proteins | is formed in nucleus, as transcription takes place

Question 27

How does splicing occur?

Answer 27

occurs via the spliceosome

Question 28

What is the spliceosome composed of

Answer 28

``` Comprised of 1) snRNPs(“snurps”),whic hare snRNAs bound to specific proteins (the U proteins); and 2) other proteins that hold the assembly together ``` U proteins recognize each other and bind to their substrates (i.e. concensus sequences of the intron) by base-pairing, using their snRNAs.

Question 29

What is the function of U proteins?

Answer 29

Sequence identification Cutting of introns Ligation of the exons together

Question 30

Describe each step how the spliceosome removes introns, and how the intron removal is precise

Answer 30

1. hnRNA (or pre-mRNA) is the substrate 2. U1 snRNA binds the 5’ side of the splice site by base-pairing snRNA to substrate 3. U2 snRNA binds to the 3’ splice site (it actually binds to the middle bit, we’re simplifying here) by base pairing 4. U6 comes in, displaces U1, and double-checks the consensus sequences is correct. Other proteins, ATP hydrolysis, and protein conformational changes form interactions with U6 and US forming an active spliceosome 5. U6 has catalytic functions: a) it cuts the 5’ splice site b) it cuts the 3’ splice site c) simultaneously joins the cut exon ends together d) releases the intron The crazy thing is that it’s actually the RNA part of U6 that does the catalysis!

Question 31

Describe what a ribozyme is | a. List all the ribozymes we come across in this class

Answer 31

are catalytic RNAs * in some eukaryotes (protozoans) as well as some mitochondria and chloroplasts, the splicing reaction is accomplished by RNA only (no protein) * Discovered in 1982 that RNA alone can act as a catalyst • it is likely that the catalytic function of the snRNPs (especially U6) is actually from the RNA portion (snRNAs) • In other words, it’s the RNA in U6 that does the catalysis, not the protein part B) ribosomes that do protein catalysis and U6

Question 32

b. Describe why RNA is hypothesized to be the first genetic carrier in the origin of life

Answer 32

NA encodes the information to make proteins, but DNA and RNA replication requires proteins – so which came first in evolution – nucleic acids or proteins? RNA can serve both as: • information storage and • catalysts • i.e. RNA has two properties needed for life. • MaybelifebeganasRNA-theso-called"RNA World". But if this hypothesis is possible, then there needs to be evidence that RNA can catalyze the synthesis of proteins – is there such evidence? • Yes,theribosome(theproteinsynthesizing machinery) turns out to be a ribozyme (stayed tuned for a later lecture!). As is U6. A hypothetical primitive cell containing RNA? The hypothesis is therefore: RNA came first, then proteins took over the catalytic functions, then DNA took over the information storage function. However, a new paper (Dec 2020) has a counter-hypothesis that both DNA and RNA operated at the beginning of life on earth. 29

Question 33

Describe what alternative splicing is, and how some exons get 'skipped'

Answer 33

different proteins from the same gene allows for different combinations of exons in the final mRNA product. Some splice sites are weak and cannot attract the U protein to form spliceosomes... so that exon gets omitted • Different tissues express different splicing enhancer proteins which will help retain the exon

Question 34

Describe how different 'splice variants' can yield proteins with different functions

Question 35

Describe the three hypotheses on why we think eukaryotic genes have introns

Answer 35

1. Higher levels of complexity due to alternative splicing: multiple potential proteins from a single gene 2. Regulatory sequences (enhancers and/or inhibitors of transcription) can be in introns 3. Introns may allow certain exons in our genome to be cut into modules or domains. These modules can be swapped between genes (exon shuffling) to make new genes (i.e. the basis of the evolutionary stuff which natural selection works upon!) • Duringmeiosis,breakpointscanoccurin introns and whole exons are transferred to a different gene -> rapid genetic evolution!! (don’t have to ‘wait’ for point mutations to provide new function) • Thismightexplainwhysomeofour genes are composites of each other.

Question 36

TFIIH

Answer 36

also acts as a helicase and unwinds the DNA strand as transcription proceeds CTD phosphorylation can be catalyzed by different protein kinases. • TFIIH acts as the protein kinase.

Question 37

Describe the (CTD).

Answer 37

RNA polymerase is heavily phosphorylated here

Question 38

Summarize the difference between PCR using a DNA template and RTPCR using a cDNA template

Answer 38

Note: we can’t prime the RT with oligio d(T) for Covid testing because prokaryotic mRNAs don’t have poly(A) tails! So instead we use ‘random hexamers’