Introduction to Genetics 304 Gene Regulation in Prokaryotes Instructor: Dr. Shelagh Campbell rta.ca

Course Topics: Transcription initiation Transcription elongation Transcription termination Translational controls Post-translational controls Complex transcriptional and translational controls (bacteriophage lambda) Coupling of transcription and translation Signal Transduction Pathways Phosphorylation Controls Protein degradation controls

Fossil remains estimated to be about 1 billion years old have been found. Bacteria have been found that live in:0 C- +120C, in acid/alkaline, able to metabolize toxins, withstand high levels of ionizing radiation. Impact on: Human diseases, nitrogen fixation, breakdown of organic material, biotechnology. Prokaryotes are highly evolved

Break: Overhead

Trp+Trp-c Experiment to Test this idea (Zamenhof and Eichorn, 1960s) Minimal media Media with tryptophan (Trp+ cells will turn off the Trp operon)

The trp-c strain is constitutively synthesizing the 5 genes in the Trp operon, all the time. This takes ENERGY. The Trp+ strain is more efficient and is therefore able to compete better for scarce resources. This seems to be a general property of prokaryotic gene regulation - if a gene isn’t needed it is turned OFF.

“Cellular economy” “Housekeeping” genes (rRNAs, tRNAs, some protein-encoding genes- stay on constitutively. Cell-cycle regulated genes (eg. for DNA replication proteins) are made at time needed. Stress-response genes (eg heat- shock genes) induced by environmental conditions. Developmental functions (eg sporulation, mating genes).

Coordinate gene expression Operons: sets of genes with related functions that are transcribed coordinately. For example the Lac operon. OR Unlinked genes that share a common set of regulatory elements. Prokaryotic genes are primarily organized in operons, unlike eukaryotes where unlinked genes are the rule.

Coupling of transcription and translation in prokaryotes due to absence of a nuclear membrane.

~100% of the prokaryotic genome is transcribed - they have very “compact” and efficiently organized genomes. In contrast, only ~3% of most eukaryotic genomes are transcribed - they have much longer regulatory regions, and an abundance of transposable elements and repetitive DNA of unknown function

RNA polymerase activities: 1) Initiation of transcription: find a promoter, bind to it, separate 2 strands of DNA 2) Transcription elongation: further unwind DNA, displace proteins that bind DNA, move along template 3) Transcription termination: recognize signals for the end of transcript and dissociate. RNA polymerase “holoenzyme” consists of a number of different protein subunits, which each contribute specific properties. VERY abundant protein (~7000 per cell)

SubunitNumberFunction a2promoter binding b1NTP binding b’1DNA binding s1initiation RNA Polymerase Holoenzyme: “core” enzyme = 2(a), 1(b), 1(b’) function: elongate transcripts by linking rNTPs to form mRNA

s factor: s factor provides specificity for RNA pol binding to promoters- without it the polymerase can only bind DNA non-specifically with low affinity. There are different kinds of s factors, most promoters we will be talking about use s 70 which is used for transcribing operons encoding metabolic genes.

How does RNA polymerase know where to start transcription - what defines a promoter? Pribnow box MODEL:

How was this model tested? 1) Genetics - selection for b-galactosidase mutants that mapped to the promoter. 2) RNA polymerase affinity measurements - ~100X variation in affinity, depending on similarity to “consensus” sequence. 3) DNA “footprinting” - form DNA/RNA complexes, map sites where proteins interact.

Translation Initiation in Prokaryotes:

Exceptions: 1) proteins with GUG or UUG initiation codons 2) secondary structure of leader sequence can affect translation efficiency: Generality: genes that are poorly transcribed are usually also poorly translated.

Key Points to remember Structure and properties of RNA polymerase. Experimental techniques for studying promoters. Optimal “promoter” sequence. Coupling of transcription and translation in prokaryotes. Optimal translation sequences. Reading review in Lewin text:  Pages  Pages