Genetics Lecture 3

What does DNA polymerase do during replication?

DNA polymerase synthesizes new DNA by adding complementary DNA nucleotides to the 3′ end of the growing strand. Therefore, DNA is synthesized 5′ → 3′. Many DNA polymerases also proofread newly synthesized DNA.

What is transcription?

The synthesis of RNA from a DNA template. RNA polymerase reads the template DNA strand 3′ → 5′ and synthesizes RNA 5′ → 3′.

What is the promoter?

A DNA sequence where RNA polymerase and transcription factors bind to initiate transcription. The promoter is upstream of the transcription start site and is generally not included in the RNA transcript.

What is the transcription start site?

The first nucleotide that is transcribed into RNA, often designated +1.

What is the template strand?

The DNA strand that RNA polymerase reads 3′ → 5′ to make a complementary RNA strand 5′ → 3′.

What is the coding/nontemplate strand?

The DNA strand whose sequence matches the RNA transcript except DNA contains T while RNA contains U.

What does upstream mean?

Toward the promoter and before the transcription start site.

What does downstream mean?

In the direction transcription proceeds, generally toward the RNA-coding region and termination site.

What is a terminator?

A DNA sequence involved in signaling the end of transcription.

What is pre-mRNA?

The initial eukaryotic RNA transcript that still requires processing before becoming mature mRNA. It may contain exons, introns, and UTR sequences.

Is hnRNA the same as pre-mRNA?

In this course context, essentially yes. hnRNA refers broadly to newly synthesized heterogeneous nuclear RNA; the portion destined to become mRNA is commonly called pre-mRNA.

What are introns?

Sequences present in the primary RNA transcript that are removed during RNA splicing.

What are exons?

Sequences that remain in the mature RNA after splicing. Important: exons are not necessarily entirely protein-coding because exons can include 5′ and 3′ UTR sequences.

What happens during RNA splicing?

Introns are removed from pre-mRNA and the remaining exons are covalently joined together.

What major processing steps convert eukaryotic pre-mRNA into mature mRNA?

5′ capping, 3′ cleavage and polyadenylation, RNA splicing, and in some RNAs RNA editing.

What is the 5′ cap?

A modified guanine nucleotide, 7-methylguanosine, added to the 5′ end of eukaryotic pre-mRNA soon after transcription begins.

What are functions of the 5′ cap?

§  Specific modified guanine added

§  Protect mRNA from being degraded

§  Helps ribosome recognize and bind mRNA later

§  Helps with RNA splicing

What nucleotide is used to add the 5′ cap?

GTP provides the guanine nucleotide that becomes the 7-methylguanosine cap.

When is the 5′ cap added?

Very early during transcription, before the entire RNA molecule has been synthesized.

What did the RNA-DNA hybridization experiment show?

Mature RNA did not pair with all regions of the DNA, causing DNA loops to form where sequences were absent from the mature RNA.

What do the loops in the RNA-DNA hybrid represent?

Introns.

Why is eukaryotic RNA processing necessary?

The primary transcript must be processed before becoming mature RNA

Why can transcription and translation occur at the same time in prokaryotes?

They are not separated by a nucleus.

Why are transcription and translation separated in eukaryotes?

Transcription occurs in the nucleus and translation occurs in the cytoplasm.

What sequence signals 3′ cleavage?

AAUAAA.

Where is the cleavage site relative to AAUAAA?

About 11–30 nucleotides downstream.

What is added after 3′ cleavage?

A poly(A) tail.

Approximately how many adenines are added to the poly(A) tail?

About 50–250 adenines.

What is the role of the poly(A) tail?

It helps stabilize and protect the mRNA.

helps translation happen efficiently

§  “Protect back end”

What is RNA splicing?

Removal of introns and joining of exons.

What is RNA editing?

Alteration of the RNA sequence after transcription.

What regions are found in a mature mRNA?

5′ cap, 5′ UTR, protein-coding region, 3′ UTR, and poly(A) tail.

What does UTR stand for?

Untranslated region.

Beginning of the mRNA, before start codon (does not code for amino acids)

Help regulate translation, ribosome get positioned so it can start translating at correct place

Is the 5′ UTR or 3′ UTR translated?

no

What marks the beginning of the protein-coding region?

The start codon.

What marks the end of the protein-coding region?

The stop codon.

What removes introns from nuclear pre-mRNA?

The spliceosome.

What is the spliceosome made of?

snRNAs and proteins.

What are the major snRNPs

U1, U2, U4, U5, and U6.

What sequence is commonly found at the 5′ splice site?

GU.

What sequence is commonly found at the 3′ splice site?

AG

What important nucleotide is found at the branch point?

Adenine, A.

What is the intron pattern to remember?

GU → branch-point A → AG.

What does U1 bind?

The 5′ splice site.

What does U2 bind?

The branch-point region.

Which snRNPs leave during spliceosome rearrangement?

U1 and U4.

What structure does the intron form during splicing?

A lariat.

What happens after the intron is removed?

the exons are joined together.

What is alternative splicing?

Different ways of joining exons from the same pre-mRNA.

What can alternative splicing produce?

Different mature mRNAs from the same gene.

What are mutually exclusive exons?

One exon or another is included, but not both.

What is alternative 5′ or 3′ splice-site selection?

Different splice sites are used, changing the length of an exon.  

Both let one gene produce multiple different RNA product and potentially different protein

What is intron retention?

An intron remains in the mature RNA.

What is alternative first exon usage?

Different first exons can be used.

What is alternative polyadenylation?

Different 3′ cleavage sites are used.

What can alternative 3′ cleavage produce?

Multiple mature mRNAs of different lengths.

Are alternative first exon usage and alternative polyadenylation mediated by the spliceosome?

no

How can abnormal splicing contribute to disease?

It can produce abnormal RNA and abnormal protein products.

What can RNA editing do to an RNA molecule?

dd, delete, or alter nucleotides after transcription.

What molecule can direct some types of RNA editing?

Guide RNA, gRNA.

What does guide RNA do?

It pairs with the RNA and helps direct where editing occurs.

What is the main function of tRNA?

to bring amino acids to the ribosome during translation.

What is the secondary structure of tRNA?

Cloverleaf.

What is the overall 3D structure of tRNA?

L-shaped.

What is the anticodon?

A three-nucleotide sequence on tRNA that pairs with an mRNA codon.

Where does an amino acid attach to tRNA?

The 3′ end.

What sequence is found at the 3′ amino acid attachment site of tRNA?

CCA.

What are some types of tRNA processing?

Cleavage, intron removal, addition of bases to the 3′ end, and chemical modification of bases.

How are rare bases in tRNA produced?

By chemical modification of standard RNA bases.

What is a ribosome made of?

rRNA and proteins.

What is the bacterial ribosome size? (prokaryotes)

70 S

What are the bacterial ribosomal subunits?

50S and 30S.

What is the eukaryotic ribosome size?

80 S

What are the eukaryotic ribosomal subunits?

60S and 40S.

What rRNAs are found in bacteria?

23S, 5S, and 16S.

What rRNAs are found in eukaryotes?

28S, 5.8S, 5S, and 18S.

What mature rRNAs come from the eukaryotic 45S precursor?

18S, 5.8S, and 28S.

How is 5S rRNA made in eukaryotes?

It is transcribed separately.

What is RNA interference?

Gene regulation using small RNAs that reduce gene expression.

What are the two small RNAs in RNA interference

miRNA and siRNA

What enzyme processes double-stranded RNA into small RNAs?

Dicer.

What complex do miRNA and siRNA work with?

RISC.

What does miRNA usually do?

Binds imperfectly and blocks translation.

What does siRNA usually do?

binds perfectly to the mRNA → causes the mRNA to be degraded

What bond links amino acids together?

peptide bonds

what are proteins made out of

amino acids

What is primary protein structure?

The amino acid sequence.

What is secondary protein structure?

Local folding such as alpha helices and beta sheets.

What mainly stabilizes secondary structure?

Hydrogen bonding.

What is tertiary structure?

The overall three-dimensional shape of one polypeptide chain. This comes from interactions between R groups/side chain

What is quaternary structure?

Multiple polypeptide chains assembled together.

What primarily determines secondary and tertiary protein structure?

Primary structure, or amino acid sequence.

What is a codon?

A group of three nucleotides in mRNA.

In what direction are codons read?

5′ → 3′.

What does it mean that the genetic code is degenerate?

More than one codon can code for the same amino acid.

What are synonymous codons?

Different codons that code for the same amino acid.

What are isoaccepting tRNAs?

Different tRNAs that carry the same amino acid.

What is the start codon?

aug

stop codons?

uga, uag, uaa