Molecular bio exam 3

Features of gene expression in prokaryotes

Gene expression in prokaryotes involves a single RNA polymerase, close regulatory elements, polycistronic mRNA (operons), coupled transcription/translation, and rare RNA processing

RNA polymerase in prokaryotes (# of kinds and # of subunits)

One kind of RNA polymerase, consisting of 4–5 subunits (two α, one β, one β′, and sometimes one ω)

Sigma factors and promoter in prokaryotes

One sigma factor binds to the promoter to recruit RNA polymerase; alternative sigma factors can be used to express specific subsets of genes during stress

Termination of transcription in prokaryotes

Transcription termination occurs via a stem-loop structure or a rho factor dependent mechanism

Repressors in lac operon regulation

Repressors like LacI bind to operators to block transcription when lactose is absent

Activators in lac operon regulation

Activators like CAP bind to enhancers to stimulate transcription when glucose is absent

Number and roles of eukaryotic RNA polymerases

Eukaryotes have up to 5 kinds of RNA polymerases (I, II, III, mitochondrial, chloroplast); Pol I synthesizes rRNA, Pol II synthesizes mRNA, Pol III synthesizes tRNA and 5S rRNA

Subunits of eukaryotic RNA polymerases

Each eukaryotic RNA polymerase contains 12–17 subunits

Role of RNA polymerase II

RNA Pol II transcribes mRNA and its CTD coordinates RNA processing

Carboxy terminal domain (CTD) of RNA Pol II

CTD is phosphorylated to convert RNA Pol II from initiation to elongation state and to recruit capping, splicing, and polyA factors

General transcription factors in eukaryotes

Large complex (e.g., TBP and TAFs) required to recruit RNA Pol II to the promoter

Mediator complex (coactivators)

Connects specific transcription factors at distant enhancers with general transcription factors and RNA polymerase at the promoter

Enhancers: role

DNA sequences bound by transcriptional activators to promote transcription

Enhancers: features

Can be close or far away (up to 100 kb), upstream or downstream, and require DNA looping to function

Specific transcription factors: action

Proteins that bind specific DNA sequences to regulate transcription of a few specific genes, usually as positive activators; repressors are uncommon

Similarities and differences in gene expression: prokaryotes vs eukaryotes

Prokaryotes: 1 RNA polymerase, close regulatory elements, polycistronic mRNA, coupled transcription/translation, rare RNA processing; Eukaryotes: multiple RNA polymerases, distant enhancers, monocistronic mRNA, uncoupled transcription/translation, extensive RNA processing

Processing of rRNA and tRNA

rRNA and tRNA are cleaved from larger pre-transcripts followed by base modifications (e.g., methylation); tRNA also receives additional CAA bases at the 3′ end

5′ cap of eukaryotic mRNA

A 7-methyl-guanosine is added to the 5′ end via an unusual 5′–5′ triphosphate linkage to protect from nucleases and serve as a translation recognition site

Polyadenylation of eukaryotic mRNA

Pre-mRNA is cleaved and about 200 adenine nucleotides are added to the 3′ end by poly(A) polymerase to protect from degradation

Splicing of introns by spliceosome

Spliceosome cleaves the 5′ splice site, forms a lariat-like loop, cleaves the 3′ splice site, and ligates exons together

Spliceosome composition (snRNA and proteins)

Composed of snRNPs (small nuclear RNAs plus proteins); U1 matches the 5′ splice junction and U2 matches the branch point

Alternative splicing

Pre-mRNAs can be spliced in more than one way to generate different proteins from the same gene

Specific transcription factors: role in transcription

Bind specific regulatory sequences to stimulate or repress transcription, usually by interacting with Mediator or general transcription factors

Domains of specific transcription factors

DNA-binding domain, dimerization domain, activation domain, and repression domain

Types of DNA-binding domains

Zinc fingers, helix-turn-helix, leucine zipper, and basic helix-loop-helix

Gene reporter assays

Method to identify regulatory sequences by linking candidate sequences to a reporter gene

Promoter deletion/mutagenesis

Method to identify important promoter regions by deleting or mutating promoter sequences

Electrophoretic mobility shift assay (EMSA)

Assay to demonstrate protein-DNA binding based on shifts in DNA mobility in a gel

DNA footprinting

Technique to identify exact DNA-binding sites by protecting bound regions from nuclease digestion

ChIP assays

Chromatin immunoprecipitation assays used to demonstrate protein-DNA interactions in cells

DNA affinity chromatography

Technique to purify DNA-binding proteins using DNA sequences attached to a column matrix

Histone acetylation

Acetylation of lysines in histone tails by HATs reduces histone affinity for DNA, relaxing chromatin for active transcription

Histone deacetylation

Deacetylation by HDACs leads to tighter chromatin packing and inactive transcription

Histone methylation

Methylation of histone tails has variable effects on transcription depending on context

DNA methylation and genomic imprinting

Methylation of cytosine bases makes DNA transcriptionally inactive; genomic imprinting uses this to turn off either the maternal or paternal allele on one chromosome

miRNA mechanism on gene expression

miRNAs bind complementary mRNA to induce cleavage/degradation or promote translational repression

Features of the genetic code

Read 5′ to 3′, three bases at a time as codons with no overlap; 1 start codon (usually AUG), 3 stop codons, and 1–6 codons per amino acid

Reading frames

There are 3 possible reading frames, but usually only one is used for a given mRNA

Codon usage bias

Unequal usage of synonymous codons for the same amino acid, influenced by %GC content and tRNA abundance

Wobble pairing

Non-standard base pairing between the 3rd position of the mRNA codon and the 1st position of the tRNA anticodon (e.g., inosine pairing with U, C, or A)

Key structural features of tRNA

70–80 nucleotides forming an L-shape with an anticodon loop and an acceptor stem containing a CCA sequence at the 3′ end

Charging tRNA with amino acids

Aminoacyl tRNA synthetase uses ATP to attach the correct amino acid to AMP, then transfers it to the 3′ OH of the tRNA using specific identity elements for recognition

Prokaryotic ribosome subunits and rRNAs

70S ribosome: 50S subunit (23S and 5S rRNAs) plus 30S subunit (16S rRNA)

Eukaryotic ribosome subunits and rRNAs

80S ribosome: 60S subunit (28S, 5.8S, and 5S rRNAs) plus 40S subunit (18S rRNA)

Prokaryotic translation initiation

30S subunit binds the Shine-Dalgarno sequence via base pairing with 16S rRNA; uses IF1, IF2, IF3 and initiator tRNA carrying formyl-methionine (fMet)

Eukaryotic translation initiation

40S subunit binds the 5′ cap and scans to find the first AUG; uses at least 11 eIF factors and a standard methionine initiator tRNA

Elongation cycle: steps

1) Decoding, 2) Transpeptidation (peptide bond formation), 3) Translocation

Role of EF-Tu/eEF1α in elongation

GTP-binding elongation factors that deliver aminoacyl-tRNA to the A site during decoding

Role of EF-G/eEF2 in elongation

GTP-binding elongation factors that drive ribosomal translocation along mRNA

Termination of translation

When a stop codon enters the A-site, a release factor binds instead of tRNA and releases the polypeptide

Role of G proteins in protein synthesis

Factors such as eEF1, eEF2, and IF2 cycle between active GTP-bound and inactive GDP-bound states to regulate steps in translation

Regulation of translation by repressor proteins

Repressor proteins can bind mRNA to inhibit translation initiation or progression

Regulation of translation by miRNAs

miRNAs can inhibit translation by binding to complementary sites on mRNAs

Global regulation of translation by eIF2/eIF2B

Phosphorylation of eIF2/eIF2B in response to stress reduces global translation initiation

Inhibition of translation by drugs

Translation can be inhibited by specific antibiotics or toxins that target ribosomes or translation factors

Protein folding: when it occurs

Folding occurs during and after synthesis, directing proteins into secondary and tertiary structures

Role of chaperones in folding

Chaperones such as Hsp70 and chaperonins assist folding, stabilize intermediates, and refold denatured proteins

Consequences of protein misfolding

Misfolded proteins can aggregate and contribute to diseases such as Alzheimer’s

N-linked glycosylation

A preassembled 14-sugar oligosaccharide is transferred to asparagine residues in the ER

O-linked glycosylation

Sugars are added one at a time to serine or threonine residues in the Golgi

N-myristoylation

Fatty acid attached to the N-terminus of a protein in the cytosol

Prenylation

Prenyl chain attached to the C-terminus of a protein in the cytosol

Palmitoylation

Fatty acid attached to internal (middle) residues of a protein in the cytosol

GPI anchor

Glycolipid added to the C-terminus of a protein in the ER

Localization of lipid-modified proteins

Lipid-modified proteins are anchored to membranes, mostly on the cytosolic face, except GPI-anchored proteins which are on the external face

Protein phosphorylation: sites and location

Addition of phosphate to serine, threonine, or tyrosine residues in the cytosol or nucleus

Regulation of protein activity by small molecules

Protein activity can change upon binding small molecules such as nucleotides (GTP/GDP) or metabolites (cAMP)

Regulation of protein activity by phosphorylation

Reversible phosphorylation by kinases and dephosphorylation by phosphatases can turn protein activity on or off

Regulation of protein activity by protein-protein interaction

Activation or inhibition of proteins via binding to another protein, such as regulatory subunits inhibiting catalytic subunits

Ubiquitin-proteasome pathway

Proteins tagged with a polyubiquitin chain are recognized and degraded by the proteasome

Autophagy

Proteins or organelles are engulfed into an autophagosome, which then fuses with a lysosome containing proteases for degradation