Readiness Quiz #2 - Rna types, RNA poly

What are the different types of RNA molecules made in a EUKARYOTIC cell?

mRNA

rRNA

tRNA

snRNA

snoRNA

miRNA

siRNA

piRNA

lncRNA

mRNA

messenger RNAs, code for proteins

rRNAs

ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis

tRNAs

transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids

snRNAs

small nuclear RNAs, function in a variety of nuclear processes, splices pre-mRNA

snoRNAs

small nucleolar RNAs, help process and chemically modify rRNAs 

miRNA

microRNAs, regulate gene expression by blocking translation of specific mRNAs, causes their degradation 

siRNA

small interfering RNAs, turn off gene expression 

piRNA

Piwi interacting RNAs, bind to piwi proteins and protect the germ line from transposable elements

lncRNA

long non coding RNAs, regulate diverse cell processes, x chromosome inactivation

THREE main RNA Polymerases for EUKARYOTIC cells

RNA pol. I

RNA pol. II

RNA pol. III

RNA polymerase I

synthesizes 5.8S, 18S, 28S rRNA

RNA Polymerase II

Synthesizes all protein coding genes, snoRNA, miRNA, siRNA, lncRNA, snRNA

RNA polymerase III

synthesizes tRNA, 5S rRNA, snRNA, genes for other small RNAs

General Transcription Factors Stepwise Biochemical Pathway

The assembly process begins when TFIID binds to a short double helical DNA sequence mostly composed of A and T nucleotides. 

This is the TATA box. 

A subunit of TFIID will recognize the TATA box called TBP (TATA binding protein) 

1. The promoter will have a TATA box.

2. TATA binding protein will recognize the TATA box and binds it. 

3. TFIIB will bind joining the TFIID complex.

4. TFIIF will stabilize and escort RNA polymerase I to the binding site.

5. TFIIE recruits TFIIH to the complex.

6. TFIIH will use energy from ATP hydrolysis to pry apart the DNA double helix at the transcription start point, exposing the strand. 

7. TFIIH phosphorylates RNA polymerase II which releases it from the general factors so that it can begin the elongation phase of transcription. 

TFII

Transcription Factor II

Why are so many GTFs needed to help RNA pol II bind to the promoter region?

The GTFs have different functions. Many are needed in order to keep RNA poly II in the right position and activated at the right time. 

They are all needed to form a complete transcription initiation complex. 

List of GTFs and what they do

1. TFIID - recognizes TATA box

2. TFIIB - recognizes BRE element in promoters, positions RNA polymerase

3. TFIIF - Stabilizes RNA polymerase interaction with other GTF 

4. TFIIE - attracts and regulates TFIIH

5. TFIIH - Unwinds DNA at the transcription point

T/F

ONLY A SMALL % OF HUMAN PROMOTERS CONTAIN THE TATA BOX

True

What are elongation factors?

They decrease the likelihood that RNA polymerase will dissociate before it reaches the end of a gene.

They associate with RNA soon after initiation and help the polymerase move along a DNA template, they are assisted by ATP. 

What direction is negative supercoiling?

TO THE LEFT 

What direction is positive supercoiling?

TO THE RIGHT

Why does DNA supercoil?

If you unwind one end of the double helix the molecule can not rotate freely. It will form a supercoil to release the tension. 

When RNA polymerase moves along the DNA it unwinds the helix ahead of itself and rewinds it behind which makes different types of supercoils.

Negative supercoiling

behind the protein, helix is unwound and is easier to open 

Positive supercoiling

ahead of the protein, helix is overwound, harder to open

What enzyme helps to relieve supercoling?

Topoisomerase

Topoisomerase temporarily breaks and rejoins the strands to relieve stress and prevent tangling

What is the purpose of pre-mRNA processing?

To convert the initial transcript into a mature mRNA molecule ready for translation

RNA processing of pre-mRNA

1. 5’ capping - modified guanine is added to 5’ end of pre-mRNA. This cap protects mRNA from degradation by exonuclease

2. RNA Splicing - Introns (non coding regions) are removed from the pre-mRNA. Exons (coding regions) are joined together to form a continuous sequence. This is done by the spliceosome.

3. 3’ Polyadenylation - Adenine nucleotides are added (the poly-A-tail) to the 3’ end of the pre-mRNA from degradation and plays a role in its transport out of the nucleus. This happens after the pre-mRNA is cleaved by an endonuclease at a specific sequence. 

Spliceosome

Recognizes specific sequences at the intro-exon boundaries

T/F

RNA processing does happens after transcription finishes its tightly coupled to RNA polymerase II elongation. The mRNA is not processed as it is being made. 

False

What is the C-terminal domain? What does it do?

CTD will recruit the right proteins at the right time. CTD will be phosphorylated by Ser5. Next the capping enzyme complex is recruited. The 5’ cap is added.

What happens to Serine 5 on the CTD?

Serine 5 is phosphorylated on the C-terminal domain of the largest subunit of RNA Pol II during transcription initiation. 

This phosphorylation lets CTD recruit 7-methylguanosine capping enzymes that deposit 7mg on the 5’ end of the mRNA.