Study Guide Chapter 8:

Why is the tern ‘junk DNA’ untenable?

 The term junk DNA is untenable because in eukaryotic genomes very little is Junk DNA
because majority of it is made up of 80% RNA product. Much of it represents pseudogenes
and retroposons.

What is the difference between an intergenic spacer and an intron?

 Intros are noncoding sequences located in the genes. Intergenic spacers are noncoding
sequences between genes.

Why does RNA generally have a shorter life compared to DNA?

 RNA can easily get picked by T2 phages when labeled uracil (u) gets pushed into
bacteria.

What would be more damaging to an organism, a null mutation in a constitutive gene, a

housekeeping gene, or a facultative gene?

 Housekeeping gene is needed for primary metabolism, facultative gene is only expressed
when needed. Housekeeping genes are more damaging to constitutive genes because it is
constantly functioning under normal and patho-physiological conditions.

Understand the three stages of transcription.

Stage 1: Initiation
- Begins in the UTR 5’ upstream
- Promoters located upstream of initiation site
- 1st nucleotide synthesized at +1 of start codon.
 Bacterial RNA polymerase (RNAP) 5 major units:
1. Beta prime: binds to DNA
- Largest unit of RNAP
2. Beta: synthesizes RNA
- 2nd largest unit
3. Alpha I & 2: attracts other components of RNAP
4. Omega: stabilizes assembly of RNAP
5. Sigma: promoter recognition factor
 RNA pol I will stop and backtrack if it detects any weak bonds due to mixmatch.
 Stage 2: Elongation

 RNA pol unwinds DNA

- Mg in RNAP binds with P of new NTP (promotes interaction with
DNA 3’ -OH)
 Synthesizes mRNA
- Compliments antisense ssDNA template
 8 bp DNA/RNA hybrid temporarily formed.
- GC rich tail is created (hairpin loop)

Stage 3: Termination

 Rho-independent (intrinsic)
- Uracil bind weakly to DNA
- RNA pol ties to back track and repair weak bonds
- Loop blocks bumping mRNA OFF RNA pol (signals release of
DNA strand).
 Rho- dependent
- Hexameric rho bind to rho-utilization site (rut)
- RNA pol slows down toward Rho-sensitive site
- Rho acts as helicase (unwinds mRNA), catches up to pause RNA
pol, and releases mRNA

Is RNA always single-stranded as Intro Bio text books depict?

 RNA is not always single stranded because RNA must become a double strand in order to
make an antisense strand.

By what mechanism does rho-independent termination work?

In Rho-independent (intrinsic) Mechanism, Uracil bind weakly to DNA. The RNA pol recognizes
the weak bond pauses the mechanism. RNA pol ties to back track and repair weak bonds. The hair
pin loop blocks bumping mRNA OFF RNA pol (signals release of DNA strand).

.
What is the role played by TBP, TATA, and the CTD tail in transcription?
TBP is a TATA binding protein that binds to the TATA box. TATA is a promoter which has a role
of determining where the genetic sequence can be read and decoded, it also determines where
transcription begins. CTD tail activates pol II which allows promoter for clearance.

What is the difference between a knock-down, a knock-out and a knock-in?

Knock down uses small interference RNA (RNAi). Knock-out/in uses crispe.

Transcription Initiation in Eukaryotes:

 General TFs bind to DNA.

- Happens before initiation
 6GTFs proteins appears
- GTF: protein transcription factors that bind to specific sites
(promoter) on DNA to activate transcription of genetic information
from DNA to messenger RNA.
- 31 MEDs recruit RNA pol II to GTFs.
- CDT ACTIVATES POL II
 TBP (TATA binding protein) TFIID bind to TATA box.
 One turn of DNA unwinds
 Antisense DNA (ssDNA) binds to RNA pol II’s active site.

Capping in RNA Synthesis

 Takes place DURING transcription

 Binds to pol II during initiation
- Interact with CTD
 Does not occur the same time as tailing
 Methyl guanine cap:
- Provide orientation
- Prevent degradation
- Guide towards cytoplasm
 4 main functions
- Blocks degeneration (Ribonucleases increasing half-life)
- Promotes intron excision (Being identifying region)
- Regulates export
- Promotes translation (Allows mRNA in interact with small ribosomal
units)

Splicing of pre-mRNA

 Introns pushed into loop cut off, exon ligated

 snRNP promotes binding to mRNA
- protein complex + U ribozymes
 U1 → GU5’ end, U2 → A site 3’ end, U4, U6, U5 ↑,
- U2 → U5 (closing loop)

Final stage of processing mRNA

 AAUAAA motif signals upstream of cleave site

 Enzyme bind to downstream GU rich region and cuts it
 Polynucleotide adenyltransferase adds lots of As.
- ATP is used as primary subtrate