Lecture 3: RNA (from dna to protein)

Question 1

Explain the central dogma of molecular biology

Answer 1

DNA —(transcription)—-> RNA—–(translation)—> protein

Question 2

What is the chemical structure of RNA/ what makes RNA different from DNA?

Answer 2

The sugar in RNA is a ribose (not a deoxyribose), so it actually has a hydroxyl on the 2’ carbon

Uracil in RNA instead of Thymine

There are still phosphodiester bonds between nucleotides to create a backbone (like DNA)

RNA is single stranded but can fold into 3D structures

Question 3

Draw the differences between ribose and deoxyribose

Draw the difference between uracil and thymine

Answer 3

Question 4

RNA has the ability to ________.

Answer 4

RNA has the ability to fold into itself

Question 5

Explain the function of the following RNA types:

1. mRNA
2. rRNA
3. tRNA
4. snRNA
5. snoRNA
6. miRNA
7. siRNA

Answer 5

1. mRNA: codes for proteins
2. ribosomal RNAs, form structure of ribosome
3. transfer RNA, critical to protein synthesis as adaptors between mRNA and AA’s
4. small nuclear RNAs, splice pre mRNA
5. miRNAs regulate gene expression by blocking translation of selective mRNAS
6. siRNAs turn off gene expression by directing degradation of selective mRNAs

Question 6

Explain the role of RNA Polymerase.

Does it require a promotor?

What is its error rate?

Answer 6

RNA Polymerase: catalyzes the formation of phosphodiester bonds between ribonucleotides

No promotor is required (more errors)

1 in 10^4 is the error rate

Note: the RNA Poly actually reads the 3 prime to 5 prime end because the pre mRNA reflects the coding strand

There is also a short region of DNA/RNA helix within the RNA Poly

Question 7

What are the signals encoded in DNA to initiate and terminate trascription?

What does RNA Poly I, II, and III do?

Answer 7

Promotor initiates transcription

Terminator (A-T forming a hairpin) stops transcription

RNA Poly I: transcribes rRNA genes

RNA Poly II: transcribes all protein coded genes

RNA Poly III: transcribes tRNA genes

Question 8

Explain how the initiation of transcription starts with RNA Poly II and its transcription factors

Answer 8

TATA box: located 25 nucleotides upstream

1. The TBP subunit of TFIID binds the TATA box, enabling TFIIB binding
2. The rest of the tx factors, incliuding RNA poly II assemble at the promoter
3. TFIIH then uses ATP to pry apart DNA at the double helix at the tx starting point
4. TFIIH ALSO phosphorylates the C terminal domain of RNA Poly II, changing its conformation so that the polymerase can release from general factors and begin elongation phase

Question 9

What are the roles of TFIID and TFIIH in transcription initiation?

Answer 9

TFIID: the TBP regonizes the tata box, and the TAF recognizes other DNA sequences near the starting point and regulates DNA binding by TBP

TFIIH: unwinds DNA at the transcription starting point, phosphorylates Ser 5 of the RNA Poly II CTD, which releases RNA Poly from the promoter

Question 10

What do transcriptional activator proteins do?

Answer 10

Transcriptional activator proteins determine the rate and pattern of transcription. They act from very very far away (thousands of nucleotides away sometimes) and they help RNA Poly, the general transcription factors, and the mediator all to assemble at the promoter.

Question 11

Explain the processing of pre mRNA to mature mRNA in Eukaryotes. What are the three things that are ESSENTIAL for the mature mRNA to get exported out of the cell nucleus?

Answer 11

1. 5’ cap
2. RNA splicing
3. poly A tail

These steps are important. If any of them don’t happen, the mRNA won’t get exported out of nucleus and into cytoplasm and therefore won’t get expressed

Question 12

Give a brief overview of RNA splicing mechanism.

Answer 12

There are several snRNAs involved, but basically U1 recognizes the 5’ splicing site, U2 recognizes the region to make a loop, the 3’ end is recognized. The intron forms a loop called a lariat, and then gets excised. The two exon regions are then united

Question 13

How can humans make so many proteins from one gene?

Answer 13

RNA SPLICING

Also the fact that we can have three different reading frames plays a role as well

Question 14

As a disease, Beta thalessemia is an issue with

Answer 14

RNA splicing

Question 15

What is the start codon? What are the stop codons?

How many reading frames are there?

Answer 15

Start codon: AUG (methionine)

Stop Codons: UAA, UAG, UGA

Three possible reading frames

Question 16

What do aminoacyl-tRNA synthases do? Do they have a proofreading mechanism?

Answer 16

tRNA synthases attach the AA to the tRNA using ATP hydrolysis.

Yes it does have a proofreading mechanism. They can remove incorrect AA’s

Question 17

Explain the basic steps on how mRNA is translated into a protein in a ribosome.

Answer 17

So you have a large ribosomal subunit and a small ribsomal subunit. There are three sites, E, P, and A.

Steps:

1. tRNA binding at the A site
2. peptide bond formation between AA in A site and the peptide chain
3. large subunit translocation
4. small subunit translocation

Question 18

What do elongation factors do?

Answer 18

Elongation factors bring tRNAs into proper position in the A site

Question 19

How does translation get initiated?

How does translation stop?

Answer 19

Translation initiation:

start codon/Met is recognized by intiation factors

Then Met tRNA binds to the P site of the small ribosomal subunit

That then binds to the 5’ end of mRNA

Translation stop:

Stops at the stop codons UAA, UAG, UGA

Release factors bind to the A site

Polypeptide is released and then ribosome dissociates

Question 20

How do antibiotics inhibit protein synthesis?

Answer 20

They block translation

Question 21

What is the role of HsP70 proteins?

Answer 21

They aid in proper protein folding (need ATP to do so)

Question 22

What are the quality control mechanisms?

Answer 22

1. 5’ cap and poly A tail
2. Exon junction complex, stimulates translation
3. nonsense mediated mRNA decay
4. protein folding and degradation