QUESTIONS:1. Name the DNA for which

transcription requires SP1.

2. What is the evidence as seen in the autoradiogram?

3. What type of transcription factor is SP1?

METHOD

1. Set up in vitro transcription reaction with DNA template, 32P-UTP, plus or minus transcription factor SP1

2. Separate transcripts by gel electrophoresis

QUESTIONS:Based on the results shown in the

autoradiogram, we see a band in lane 1 (from transfection with plasmid 1) with but not in lane 2 (from transfection with plasmid 2).

1. What does the band in the autoradiogram represent?

2. What do these results tell us about the nature of the extra piece of DNA included in plasmid 1 and absent in plasmid 2? (What is the “name” for this extra piece of DNA?)

Lecture 9

Transcription Termination,

mRNA processing, & post-transcriptional control

Reading:

Chapter 12

Molecular Biology syllabus web site

Transcription termination

• Several mechanisms exist to regulate the termination of transcription in bacteria and eukaryotic cells

• In bacteria, the two principle mechanisms involve RNA polymerase and one of these also requires the termination factor Rho

• In eukaryotes, the mechanisms for terminating transcription differ for each of the three types of RNA polymerase

Rho-independent termination occurs at characteristic sequences in E. coli DNA

Premature termination by attenuation helps regulate expression of some bacterial

operons

Mechanism of attenuation of trp-operon transcription

Rho-dependent termination sites are present in some -phage and E. coli

genes

Figure 11-4

• The Rho factor is a hexameric protein around which a 70- to 80-base segment of the growing RNA transcript wraps

• Rho then moves along the RNA in the 3 direction until it eventually unwinds the RNA-DNA hybrid at the active site of RNA polymerase

• Whether transcription is terminated or not depends on whether Rho “catches up” to RNA polymerase

• Rho-dependent sites have no clear consensus sequence and Rho-dependent termination operates at relatively few operons

Three eukaryotic RNA polymerases employ different termination

mechanisms • RNA polymerase I is terminated by a mechanism that

requires a polymerase-specific termination factor, which binds downstream of the transcription unit

• RNA polymerase II is terminated in a region 0.5-2 kb beyond the poly(A) addition site, and termination is coupled to the process that cleaves and polyadenylates the 3 end of a transcript

• RNA polymerase III is terminated after polymerizing a series of U residues

Transcription of HIV genome is regulated by an antitermination

mechanism

Processing of eukaryotic mRNA

The 5-cap is added to nascent RNAs after initiation by RNA polymerase II

Pre-mRNAs are cleaved at specific 3 sites and rapidly polyadenylated

During the final step in formation of mature, functional mRNA, introns are removed and

exons are spliced together

Splicing occurs at short, conserved sequences

Consensus sequences around 5 and 3 splice sites in vertebrate pre-mRNA

Analysis of RNA products formed in an in vitro splicing reaction

Splicing proceeds via two sequential transesterfication reactions

Small nuclear RNAs (snRNAs) assist in the splicing reaction

Self-splicing introns-evolutionary models of trans-acting snRNAs?

Other post-transcriptional regulatory mechanisms

• Alternative splicing (e.g. ion channels affecting auditory cells; affecting wiring/neuronal connections in the brain)

• mRNA 3’ ends that target mRNA to cytoplasmic location (resulting in protein gradients across cell)

• mRNA stability affected by 3’ untranslated sequences• Regulation of antisense transcripts and siRNA• RNA editing (common in mt in protozoa/plants and

chloroplasts; rarer in higher eukaryotes)• Post-transcriptional modification (e.g. tRNA)• Translational activation of mRNAs (cytoplasmic

polyadenylation of stored mRNAs with short polyA tails- induced upon fertilization of xenopus oocytes or synaptic activity in neuronal dendrites)