Notes

MBB 222

Lectures 25 & 26

>Genes can be expressed with different efficiencies, this can be regulated by the production of RNA

RNA vs DNA:
>RNA has ribose rather than Deoxyribose
>RNA has Uracil rather than Thymine
>RNA is usually single stranded and highly reactive because of exposed bases and the 2'OH group on the ribose.

>RNA can base pair within itself for sec. structures (WC and non-WC bp)

Types of RNA:
>mRNA->'M'essenger RNA (code for proteins)
>rRNA->'R'ibosomal RNA (catalyze protein synthesis)
>tRNA->'T'ransfer RNA connects helps connect free base pairs during protein synthesis
>snRNA->'S'mall 'N'uclear RNA, variety of processes
>snoRNA->'S'mall 'N'ucleOLAR RNA, used to produce and modify rRNA
>scaRNA->'S'mall 'C'ajal RNA, used to modify sn/snoRNA
>miRNA->'M'icro RNA, regulate gene expression by blocking translation of certain mRNA
>sirRNA->'S'mall 'I'nterfering RNA, turn off gene expression by degrading mRNA and creating compact chromatin structures

>The DNA Coding strand is the EXACT same as the RNA transcript except that in the RNA sequence Uracil is used in place of T!

>Prokaryotic transcription->since there is no nucleus in Pro. the mRNA is accessible immediately and transcription and translation occur in a 'coupled' fashion.
>Eukaryotic transcription->mRNA have to be processed before leaving the nucleus so the two processes are separate

"Processing of mRNA in Eukaryotes":
>5' cap
>3' polyA tail
>Splicing to remove introns, and have a continuous coding region

In bacteria the most critical part for gene control is transcription:
>mRNA synthesis
>mRNA degradation

Elements of gene control in Eukaryotes:
>transcription initiation
>nuclease degradation
>transcription termination

>Transcription requires dsDNA, ribonucleotides (NTPs) and MG2+ ions

Transcription of by RNA polymerase in E. Coli:
>17 bp of DNA are unwound in a "Transcription bubble" the DNA rotates to allow the bubble to move 5' to 3' as the RNA sequence is being transcribed; NTP channels allow NTPs to enter during transcription and the RNA/DNA hybrid breaks off the RNA transcript.

>Promoters are upstream of the coding sequence and are recognized by RNA polymerase to start transcription, downstream are positive numbers and upstream from the initiation site are negative numbers, similarities occur b/w organisms at -10 and -35.

Bacterial RNA polymerase sequence:
>INITIATION: RNA pol. holoenzyme & sigma factor scans the strand until it finds a promoter
>Polymerase unwinds the DNA and starts to transcribe it, after about 8 nucleotide the sigma factor is released and transcription occurs at a more rapid pace. (Sigma factor gives the polymerase specificity for DNA of its own species)

Prokaryotic Elongation:
>no sigma factor needed
>moves 50bp/second
>no editing during polymerization

>Errors in RNA are not critically important because they do not pass their info onto daughter cells and also are not the long term storage of information.

GENE expression:
>housekeeping genes are expressed at all times
>THere are inducible and repressible gene products, this is done by RNA interactions with the promoters.
>Promoters can be turned on or off by areas near them.

>In Bacteria genes with similar proteins are encoded together in an operon, these are transcribed together on a polycistronic mRNA!
>Transcription of operons are induced or repressed by effector molecules->allosteric regulation
Two types of effectors:
>inducers - decrease the affinity of the repressor for the operator
>co-repressors - increase the affinity of the repressor for the operator

The Lac Operon:
>Beta-galactotidase-> gene z (hydrolize lactose to glucose and galactose)
>Galactoside-permease-> gene y (transport lactose across the bacterial membrane)
>Thiogalactoside transacetylase-> gene a (may be used to export toxins made from the permease)

Negative regulation of the lac operon:
(a) When there is no lactose present the lac repressor binds to the operator and blocks transcription via RNA polymerase
(b) When lactose is present the side product allolactose is produced (by already present beta-gal.) and it binds to the repressor, there is a resulting conformational change that causes the repressor to be removed from the operator and transcription of the lac operon can occur.

>IPTG is often used experimentally to induce gene expression. The lac promoter (P) and operator (O) are inserted into the plasmid and the gene of interest is placed after (“downstream of”) the lac promoter/operator (in the MCS) so that its expression is under control of that promoter and regulated by the repressor and IPTG.
>thus, one can trigger the expression of the target gene, simply by adding IPTG to the medium

>DNA foot-printing (the lac repressor protects the operator from digestion)
>The Lac repressor binds DNA via a helix-turn-helix motif

KNOW** positive regulation of lac operon with crp and camp.

>Trp stuff is easy, the repressor can't bind to the operator so mRNA transcribes the five TRP structural genes and they all work to make tryptophan which in turn binds to the repressor which conformationally changes to fit on the operator, to block further transcription.