Lecture 8 &9- Gene Expression 1 and 2 Flashcards
Prokaryotic mRNAs
Left of AUG codon is a rbi, where 30s ribosomal subunit binds. 30s unit can also come in in the middle of an mRNA unit at next site and start translating- eukaryotes cannot do this. 50s comes in next, binds to 30s. This mRNA has two cisterns; bicistronic. This means two regions that code for protein. Each region contains a series of codons that when translated produce a protein. There is a terminal codon at end of region. Ribosome can either stop or fall off, or continue translating along mRNA until it comes to next AUG codon.
Eukaryotic mRNAs
40 and 60s (80s) instead of 30/50. Almost all eukaryotic mRNAs cistronic. 3 different features: 5’ cap is an unusual nuc structure where nucs are methylated; polyA tail is a string of a’s; almost all monocistronic. The first aug rule: ribosome cannot join in the middle, all mRNAs to be translated by euk must adhere. Only one codon can be accessed or translated on monocistronic rna. Leaky scanning- exception.
Starts with 40s subunit binding to 5’ cap. 40s translocates along ran toward internal sites (scanning) w/o 60s ran to find first aug. once found, 60s joins 40s. first aug translation begins, terminates at termination region at end of coding region. ribosome falls off.
Gene expression for eukaryotic DNA viruses
promoter for each gene (adeno, herpes, sv40, poxviruses). These viruses have multiple promoters to solve this. Creates monocistronic mRNAs RNA splicing (same viruses): RNA splicing takes advantage of cell machinery in nuc, pox viruses replicate in cytoplasm. Splice donor must be to the left of the first aug so that the first aug is spliced out and the second aug becomes the first aug. Splicing is not 100%, which is good because then only gene 2 would be translated.
Gene expression for eukaryotic plus-strand RNA viruses
Picornaviruses (polio, rhinovirus), flaviviruses (hep c, west nile virus, dengue, zika)
Cannot regulate relative abundance with this scheme. not energetically favorable but it solves the first aug problem. Has 5’ and poly a tail, goes through translation give viral polyprotein, proteolysis (proteolytic cleavage) to give individual protein units. Just have one aug: produces one long polypeptide chain. Make more capsid proteins that enzymes (which can be recycled). Protease is embedded within polyprotein itself. W/in region, active proteins. it can bind and cleave. Viral polyprotein has at least some regions which fold up normally. Sometimes host is involved in cleaving, mostly not.
Gene expression for eukaryotic plus-strand RNA viruses- Toga viruses (semliki forest virus/SFV, sindbis (enveloped viruses)
Have first aug, and an aug that is not accessible. Genes for enzymes after first aug, genes for structural protein after middle aug. Makes polyprotein; then cleaved into individual polyproteins (similar to before). One protein is the viral replicase (ran pol). makes viral replicase so that it can access second aug. This species can regulate: makes a lot more of structural proteins than enzymes.
Plus strand RNA viruses- Corona viruses (mouse hep virus, SARS, MERS)
have a plus RNA with 7 genes, 1st gene codes for polymerase. (viral replicase/transcriptase). This is b/c can’t access the other codons (2-7). 5’ end has leader seq and cap, others do not have cap initially, just aug.
Makes an antigenome, then prod series of RNAs, first w/ 2-7, then 3-7, etc.
get nested set of plus mrnas all with same 3’ end and all with same short leader seq on 5’ end.
double check: does OH regulate which aug to start coding at, or is it random?
Gene Expression for eukaryotic minus strand RNA viruses
Rhabdoviruses (VSV, rabies), paramyxoviruses (measles, sendai) and filoviruses (ebola)
vdv has 5 genes, transcriptase associated with ran w/in virion particle. transcriptase also adds 5’ caps and poly a tails, starts/stops/starts. Have monocistronic mRNAs (+) capped with poly a
translate to individual viral proteins
Minus-strand RNA viruses- Orthomyxoviruses (influenza)
cap snatching: all caps derived from host cell
Have genome RNA - (8 SEGMENTS). Solves by having each RNA reg correspond to a single gene, each transcribes monocistronic DNA. In nucleus of infected cell, virion transcriptase; 5’ ends taken from cell mRNAs. get monocistronic mRNAs with poly a tail and 5’ caps. Segmentation allows virus to adhere to first aug rule; each segment is a gene. can actually produce more than just those 8 proteins.
most RNA viruses replicate in cytoplasm of cell, except influenza which replicates in nucleus, means it can expand on gen material using splicing
poxvirus = dna virus, replicates in cytoplasm.
Initiation of synthesis of influenza mRNAs (cap snatching in nucleus)
endonuclease activity cleaves RNA without any seq specificity; steals piece of RNA from host cell to use as primer to synth own rna. does not have much seq specificity. Transcriptase has associated nuclease, 10-13 nuc from 5 end, will cleave. Uses 3’ end of minus viral RNA segment as primer to extend and transcribe almost full length of segment. get monocistronic mRNA with 5’ cap
Splicing of mRNAs from segments 7 and 8 (orthomyxovirsuses (influenza))
for 7 and 8, initial mRNA produced is spliced to produce two proteins. Coding region for these two overlap. Whenever this happens, the two coding regions just be in different reading frames. Splice donor to left of gene one, and to left of gene 2. Splicing not 100%, so both gene products can be made.
Gene expression for ambisense RNA viruses
Bunyaviruses (hanta virus) and arena viruses (lymphocytic choriomeningitis virus/LCM)
Why can’t plus genome itself act as mRNA? no 5’ cap
Sometimes just two segments, sometimes more. Gene 2 synthesized first because it is complementary to mRNA. Minus sense res through early transcription by virion transcriptase, and then translation. This makes the transcriptase/replicase, which goes back and makes plus strand’s antigenome, then plus rna w/ 5’, then makes proteins. Plus sense has to go through process of making antigenome, then late transcription (plus mRNA with 5’ cap), then translation (late proteins). Transcriptase carried w/in virion; starts at 3’ end and turns it into plus.
Gene expression for double stranded RNA virus
Reovirus and phi6
segmented ds RNA genome (reovirus: 10 segments, phi6: 3 segments)
similar to vsv, gene sized monocistronic mRNA
virion transcriptase gives monocistronic mRNAs (+) (reo has caps, no poly a tail on either res or phi 6 mRNAs). translation of these (each has aug) gives viral proteins. each set produced from monocistronic rna.
reovirus= conservative
have ds dna, looks like transcription. nascent chain grows and is displaced, left with two parent strands still attached and nascent chain separate.
phage phi6 = semiconservative
ds dna, nascent chain remains base paired with template strand throughout entire synth of chain. newly made chain bped, original lower strand is now alone. only one of the parental strands is conserved.
Gene expression for bacteriophage T4
Have early middle and late. A few genes are transcribed, one of the genes produced in early modifies system so now middle rna starts getting produced. one of the middle products codes for a protein that starts turning on production of the later products. eventually get lysis
