1. Name several processes by which the level of protein may be regulated in the cell.

b. Transcription. Particularly the regulation of mRNA synthesis at the level of initiation, i.e., as emphasized in class

c. Post-transcriptional mRNA processing, including conversion of intron containing mRNA to translatable RNA in eukaryotes. In addition, mRNA degradation, conversion of polysistronic mRNA to individual cistronic mRNAs, and other events like "RNA editing". Some of these mechanisms will be discussed in other courses.

d. Translation. Translation, like transcription, may be regulated at the levels of initiation and elongation. RNA-binding proteins are an important component of gene regulatory circuits in biology. Many examples of translational regulation will be discussed in the Second Semester course, Biochemistry 718.

e. Post-translational protein modification. Processing of "polyproteins" by proteases, cleavage of proteins (such as removal of "signal" peptides), phosphorylation-dephosphorlation, acetylation and addition of carbohydrate and lipid moieties are some of the mechanisms that affect function and stability of the protein products of genes.

2. Give examples of cis-acting and trans-acting genetic factors that control transcription.

cis-acting: promoter, operator, silencer, enhancer

trans-acting: RNA polymerase, repressor, TATA Binding Protein, TFIIB, CAP, tryptophan.

You should be able to predict the effects of such factors.

3. Explain regulation of the E. coli lactose operon.

You should be able to summarize the steps involved in activation and repression of this operon in response to the type of sugar available as carbon source for this organism.

4. Explain how the levels of tryptophan in an E. coli cell regulate the biosynthesis of this amino acid through transcriptional repression-derepression and attenuation of the tryptohan operon.

5. Explain the probable roles of the (a) major and minor grooves, (b) side-groups of the purine-pyrimidine base pairs, and (c) deoxyribose phosphate backbone of the promoter in binding the E. coli RNA polymerase (RNAP) holoenzyme.

Unlike "core" RNAP, which binds DNA independently of the nucleotide sequence, the holoenzyme binds a specific sequence. Sequence-independent binding DNA binding proteins like RNAP core and the histones of eukaryotes generally use electrostatic interactions with the phosphate backbone. Histones are bound to the minor-groove side of DNA thus, permitting access to the major groove by sequence-specific DNA binding proteins. E. coli RNAP holoenzyme is able to recognize promoter sequences through the DNA major groove. Sequence-specificity is imparted to the RNAP by sigma factors. The s ⁷⁰ factor binds the -35/-10 region of the promoter only if it is associated with RNAP core. In E. coli there are different s factors for different classes of promoters. Some s factors bind promoter DNA sequences independently of their association with RNAP and, under the appropriate conditions, recruit RNAP to promoters, i.e., in an analogous fashion to the recruitment of eukaryotic RNAP to promoters by transcription factors. Geometry of the base-pair side-groups in the major groove determine specificity to the amino-acid sequence of the DNA-binding domain of the protein. Many proteins interact with their specific DNA sequences through hydrogen bonding between the side groups of the DNA base-pairs and side-groups of the amino-acid sequence that binds the DNA. The a -helix of proteins fits just right in the major groove of B-form DNA and is often the bearer of the nucleotide-sequence-specificity of the protein. You should be familiar with the different protein "motifs" that have been implicated in recognition of specific DNA sequences in prokaryotes and eukaryotes.