512 Exam 2 Lecture 9

What direction on the template strand does an RNA polymerase travel?

What direction on the template strand does an RNA polymerase travel?

3’ to 5’

What direction are nucleotides added?

5’ to 3’

What is unique about eukaryotic transcripts from most RNA virus transcripts?

Human transcripts have introns and exons (introns must be removed and this occurs in the nucleus)

What features are needed at the 5’ and 3’ end of a eukaryotic mRNA for it to be translated?

5’ cap and 3’ polyA tail

What is meant by leaky scanning and ribosomal frameshifting?

Leaky scanning: when translation is initiated at second AUG and not the first AUG which makes for a different open reading frame

What are the similarities and differences between leaky scanning and ribosomal frameshifting?

Similarities:

• multiple/completely different proteins from 1 transcript (more genetic material in one space)

• make proteins in different reading frames

Differences:

• Leaky: ribosome misses first AUG and then goes to the next one (shorter version of protein)

• Ribo: after translation has already started, same N-terminus of the og protein

What are the 3 types of genomes that RNA viruses can have?

1. Positive sense RNA

2. Negative sense RNA

3. dsRNA (+/-)

What are the size ranges of RNA viruses?

6 kb to 41 kb

Are dsRNA viruses segmented, non-segmented, or both? How about (+) RNA viruses? (-) RNA viruses?

dsRNA viruses: exclusively segmented

(+) RNA: predominantly non-segmented

(-) RNA: either

Why must an RNA virus encode for an RNA-dependent RNA polymerase?

Virus life cycle is in the cytoplasm, don’t have access to host polymerase, so they need to encode for their own

Define the difference between replication and transcription?

Replication: making exact copies of the genome to be packaged into new virions

Transcription: making an RNA to be translated into protein (or has some other function)

Why in some cases can replication and transcription processes overlap for RNA viruses?

Different RNAs are made for transcription/replication, in others the genome RNA can also be used as an mRNA

RNA viruses use the same enzyme for both

In what class of RNA viruses do replication and transcription processes commonly overlap?

(+) sense

Do RNA viruses use the same enzyme to replicate and transcribe RNA? Do they use the same complex of proteins?

Yes; no

What are the signals that in the genome help initiate replication/transcription?

Transcription:

• 3’ end

• production of leader RNA

• transcription start site (aka initiation sequence)

Replication:

• polymerase must ignore termination signals

Do RNA viruses require a cap and a polyA tail?

No

Of the viruses we discussed, which ones have caps and polyA tails, which are missing one of these?

Picornavirus (do not have cap)

Flavivirus (do not have polyA tail)

Alphavirus (has both)

Coronavirus (has both)

What are the basic properties of (-) RNA viruses? Are they single- or multisegmented?

• enveloped

• form helical nucleocapsids

• do not make polyproteins

• replicate in cytoplasm

• single and multisegmented genomes

What non-structural protein must be in the virion for (-) RNA viruses?

RNA polymerase

What are some common (-) RNA viruses?

Rhabdoviridae

Paramyxoviruses

Filoviruses

What are the five proteins in all monopartite (-) RNA viruses?

N- nucleoprotein

P- phosphoprotein

M- matrix protein

G- glycoprotein

L- large protein (polymerase)

Are the proteins (monopartite) made from one transcript or multiple transcripts?

5 separate mRNAs created; 1 for each protein (because of stuttering process)

In what order are these proteins made?

N > P > M > G > L

Which proteins comprise the RNP complex?

N

P

L

Circular

Describe the process of rhabdovirus transcription.

1. transcription begins at the 3’ end of the genomic template RNA

2. begins with the production of leader RNA

3. RNP continues moving along genome without transcription until it encounters a transcription start site

4. after initiation a cap is added to mRNA

5. when RNP meets a termination sequence it will add polyA tail

6. mRNA is released (free for for translation)

What is the leader RNA in Rhabdoviruses?

• 49 nucleotide RNA

• non-capped

• no polyA tail

• no known function

When Rhabdovirus RNA polymerase encounters a termination sequence, what are its two choices?

1. dissociate from RNA, go back to the beginning and start over

2. move along genome until it encounters another initiation sequence (stuttering)

What is the stuttering mechanism?

idea that polymerase at that point can “choose” either dissociate from the RNA or chug along and hit the start site and start transcribing the next gene

What is EIS?

E- end domain sequence

I- RNA skips dinucleotide

S- RNP reinitiates at start sequence 3’-UUGUC-5’

How is rhabdovirus transcription regulated?

RNP must switch from transcription to replicating viral genome

How is this regulation achieved?

Anti-genome: complete copy of genome complementary to genome RNA

What must happen for Rhabdovirus (-) RNA to be replicated vs transcribed?

polymerase must ignore the termination signals (so much end protein built up, just bypass the signal)

Do Rhabdovirus mRNAs encode for one or multiple proteins?

5 proteins

Is Rhabdovirus RNA replication symmetric or asymmetric?

asymmetric

What does asymmetric mean?

(-) RNA made at massive abdundances much more than the (+) genome

Which strand is made in abundance?

(-) RNA >> (+) anti-genome