Mcgill Bio 200 - Lecture 18

Front 1

Agarose acts like a sieve to separate DNA & RNA according to ____.

Back 1

Size

Front 2

Why must nucleic acids be transferred to solid state support? What supports do you use for DNA? RNA?

Back 2

If left on agarose gel, the molecules would diffuse, losing their diagnostic signature. DNA support uses nylon and nitrocellulose. RNA just uses nitrocellulose.

Front 3

What would occur if you didn't denature RNA before performing a Northern Blot?

Back 3

RNA would remain twisted and double stranded and would you would not be able to properly characterize it.

Front 4

Which sequences (strands) on the blot are recognized by autoradiography?

Back 4

Complementary sequences.

Front 5

Southern Blot is for DNA to ___, whereas Northern blot is for a gene's RNA with either a gene's ___ or ____.

Back 5

DNA. RNA or cDNA.

Front 6

How do RFLPs arise?

Back 6

These nucleic acid fragments arise when due to restriction sites being created or destroyed by mutations. Depending on which sites are functional, the RFLP will be made but cuts at various places on the gene.

Front 7

What do you use to keep RNA denatured? (Hint: 'formal...')

Back 7

Formaldehyde

Front 8

The length of a gene is affected by alternative splicing. If we confirm this by nucleic acid analysis, this would be a _____ observation.

Back 8

Qualitative

Front 9

True or False: Nucleic acid hybridization can tell us what stage of development a tissue is in.

Back 9

True. Abundance and size of nucliec acid can indicate such things.

Front 10

List the RNA polymerases in INCREASING order of inhibition by the mushroom toxin alpha-amanitin.

Back 10

RNA pol I (not inhibited), RNA pol III (moderate), RNA pol II (strongly inhibited)

Front 11

Which stage of transcription is strongly regulated?

Back 11

Initiation

Front 12

Name three places where transcription occurs in eukaryotes. (Hint: these places have genes)

Back 12

Plastids, mitochondria, nucleus.

Front 13

What does RNA pol I primarily transcribe? What are 28S, 5.8S, 18S examples of

Back 13

ribosome components and ribosomal precursor RNA genes in the nucleolus.
Those are examples of ribosomal precursor RNA genes.

Front 14

What does RNA polymerase II primarily transcribe? True or false ; RNA pol II transcribes for splicing snRNAs (U1- U5) and other functional RNAs.

Back 14

all protein encoding genes. TRUE.

Front 15

What does RNA polymerase III transcribe?

Back 15

ribosomal RNA genes outside the nucleolus (5S), tRNA genes and RNAS including U6 for splicing.

Front 16

What are some similarities between the three RNA polymerases? (Hint structure, core ___, and the important protein of TFIID).

Back 16

Multimeric protein complexes (>10), core subunits are similar, and TBP optimizes transcription of each class of gene.

Front 17

The TATA box is located roughly at -35 and consists of ______. It serves to positions and orient RNA _____

Back 17

As & Ts. It orients polymerase.

Front 18

The Tata Box binding protein (TBP) is part of the general transcription factor ____. It has conserved C terminal domain which binds to the ____ groove of the TATA box.

Back 18

TFIID, minor

Front 19

The TBP is similar to a sigma factor in bacteria in that it..(think specializes). Do TATA-less sequences require TBP for initiation?

Back 19

Confers specificity to RNA pol II. YES!

Front 20

What is unique about the promoter in RNA pol III?

Back 20

The promoter element is located within the gene that gets transcribed.

Front 21

List the correct orders of general transcription factor association for RNA polymerase II preinitiation complex in vitro.

Back 21

TBP (of TFIID) binds, then TFIIB, then RNA pol II and TFIIF associate. TFIIE (4 subunit factor) associated then TFIIH associates.

Front 22

What are uniques traits of RNA polymerase II

Back 22

Only pol II requires ATP hydrolysis (due to TFIIH factor's helicase ability). RNA pol II has a Carboxy Terminal Domain (big giveaway!)

Front 23

Why is TFIIH helicase activity important?

Back 23

It unwinds the DNA strands to form open complex (transcription bubble) RNA pol II can work in.

Front 24

In vitro, phosphorylation of CTD by TFIIH protein kinase causes dissociation of all but which general transcription factors?

Back 24

TBP

Front 25

What is the difference between in vivo and in vitro regarding the initiation complex. What will an in vivo RNA pol II transcription complex resemble in prokaryotic cells?

Back 25

In vitro, most general transcription factors dissociate. In vivo, we are not sure which factors dissociate. It will resemble the holoenzyme complex.

Front 26

What nucletide repair is TFIIH involved in? What is evidence of TFIIH's duality?

Back 26

Nucleotide excision repair. the most rapidly repaired regions are the ones transcribed.

Front 27

RNA polymerase I: Talk about core element, the Upstream element, and what factors affect them.

Back 27

Core element : -40 to +5. Essential for transcription. Core factor (containing TBP) associated growing assembly)
Upstream Element: -155 to -60. Enhances transcription efficiency. Upstream Activating Factor recognizes the upstream element and commences assembly in vitro.

Front 28

Which RNAs make up the greatest amount of RNA transcribed? Which is less than 10%

Back 28

tRNA and 5S rRNA. mRNA is <10%

Front 29

What is so weird about RNA pol III?

Back 29

Is has promoters in the transcribed regions, conserved internal promoter elements. The A box and B box of this element also encode elements of tRNA.

Front 30

What does RNA pol III control transcription of?

Back 30

Transcription of tRNA and 5S rRNA.

Front 31

The C box in genes transcribed by RNA pol III acts as a promoter for _____

Back 31

5S gene transcription.

Front 32

Whats the sequential assembly of general transcription factors in RNA pol III?

Back 32

TFIIIC (w/TFIIIA in 5S), TFIIIB, THEN pol III.