Genetics: E3-A

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Transcription: promotor recognition and initiation details

RNA polymerase binds to promotor region at 3' end of template (usually upstream of coding region). It locally unwinds the DNA there. Since it doesn't need a primer, it just starts adding free nucleotides to the template strand.

Transcription: chain elongation details

RNA polymerase addes to the 3' end of the transcript following rules of base pairing except using U instead of T when pairing with A. **requires Mg, energy from splitting triphosphate nucleotides.

Transcription: chain termination details

At 5' end of transcript. RNA polymerase stops when it hits inverted repeats to tell it to let go. Inverted repeats (palindroms), which may form a hair pin loop by complementary base pairing, is a form of "self-termination" (E.coli). Or, termination can occur via enzymes that come and cut like endonucleases in humans. <-- called 3' cleavage.

What are the basic steps of transcription?

1.) promotor recognition and intiation. 2.) chain elongation 3.) chain termination.

If promotor sequences are similar to the consensus sequence, what happens? What if they are different?

If similar, transcription can begin more often. If difference, transcription occurs slowly and will be iniated less frequency. ***simliary = strongly expressed. Different = weakly expressed.

consensus sequence

most common sequence or "motif" of all the protomor regions. ***The closer a promotor is to the consensus sequence, the "stronger" the promotor is and the more likely it is to be transcribed over those that are less similar to the consensus sequence. Ex. TATA box in eukaryotes and -35 and -10 promotor sequences.

What is a promotor region

nucleotide sequence of 20-200 bP long; intial binding site for RNA poly and transcription iniation factors. Somewhat conserved among genes and to a lesser degree b/w species. Promotor regions are found on both DNA strands. *determines where transcription begins. **Promotos are never transcribed or translated; they are purely REGULATORY.

When does complementary base pairing occur?

transcription to from RNA, RNA processing to form mature protein, during translation b/w codon and anticodon, during post-transcriptional and post-translational processing

What are DNA binding proteins

recognize double or single stranded DNA; recognize specific nucleotide sequences.

How many mRNA can we make from a single gene?

many. In prokaryotes, transcription is coupled with translation such that translation occurs even before transcription occurs. This doesn't happen in eukaryotes however.

Which strand is the template strand?

It is random which one is the template strand from gene to gene however, it is mostly constant for each individual gene.

Which way does RNA polymerase move during transcription? In which direction is the mRNA produced? Which is the growing end?

3' to 5' along template strand; the mRNA transcript is created in the 5' to 3' direction with the 3' end being the growing end

Things about RNA polymerase that you should know

doesn't need a primer. Locally unwinds DNA by itself. Goes by normal complementary base pairing but instead of adding a T for A, it adds uracil. Reads template strand from 3 to 5'. Less sophisticated proofreading mechanisms than DNA pol in DNA synthesis which is why RNA virsues are more abundant/bad.

Which is more prone to errors: DNA synthesis or transcription?

Transcription because RNA poly has less proofreading capabilities than DNA poly.

Classes of RNA

mRNA - product of transcription (final transcript for prokaryotes; processed transcript in eukaryotes). Functional RNAs include tRNA, rRNA, and small RNA.

Function of tRNA

bring ammino acids to ribosome

Function of rRNA

structural and catalytic components of ribosomes; large and small subunit only come together for transcription.

small RNA functions

3 types: small nuclear, small cytoplasm, and micro. They are used for making up splicesome, directing protein traffic in cytoplasm, and transcription/trans reguation, respectively.

Is it possible to have differen promotors for different stages of life?

yes. We see this in larval and adult transcription. Different promotor for larval and adult. Get different pre-mRNA but same mRNA. Another way to increase genetic variation.

Where are termination sites usually located?

located such that transcribed regions DO NOT overlap.

Eukaryotic RNA processing

1.) Addition of a 3' poly-A tail and 5' cap. 2.) intron excision 3.) exon splicing

5' cap

addition of a 7-methylguanosine (modified guanine), linked by 3 phosphates, prevents enzymatic degradation of transcript, recognized by ribosomes.

3' poly(A) tail

addition of up to 200 adenine molecules to the 3' end. Occurs downstream of AAuAAA (poly adenylation signal).

Do eukaryotic pre-mRNA have untranslated regions?

Yes. They are called the 3'UTR and 5'UTR.

When is the 5' cap and the 3' poly(A) tail added during eukaryotic transcription?

5' cap is added to the pre-mRNA as transcription is occuring. 3' poly-A-Tail is added once transcription has occurred. Both the 5' cap and 3' poly-A-tail occur before splicing occuring/mRNA results.

What is the purpose of the polyadenylation signal?

Directions addition of poly (A) tail. It is a conserved sequence of bases: AAUAAA.

Intron excision: how does it happen?

Done via splicesomes/5 snRNPs. All introns have 5'GU and 3'AG recognition sequences (Gu-AG rule) which tells splisosomes where to cut and a middle branch point A. The 2'-OH from branch point A helps to cut RNA at 5' splice site. cut 5' end is linked to branch point A. Exons ligate and spliceosome released. Introns excised are degraded. Branch point adenines are conserved. **Intron excised as lariat.

Are all introns removed before relase of the mRNA from the transcription complex?

yes. In eukaryotes, transcription and RNA processing are coupled. **mRNA processing begins before transcription is complete!

What do snRNPs do?

make up of proteins and small RNAs that recognize splice junctions. They make up spliceosomes.

How does termination of copying differ for prokaryotes and eukaryotes?

For prokaryotes, copying goes past the translation termination codon (into the 5' UTR) and continues until a specilized region that terminates transcription (inverted sequences). For eukaryotes, copying continues past the conserved AAUAAA sequence (polyadenylase signal), where transcription terminates.

What happens if there is a mutation or something that causes the spliceosome to be even 1 nucleotide off of the actual splice site? (i.e. what happens when cutting doesn't occur by the GU-AG rule?)

BAD bad things. Cancer. Disease. Pontacerebellar hypoplasia is due to a mutated splicesite which leads to abnormally edited RNA transcript.

What is the average human gene made up of mostly: introns or exons?

INTRONS mostly. Only a few exons.

T or F: With eukaryotes and prokaryotes, simple organisms don't have many introns while more complexed organisms have more introns.

F. Only holds true for eukaryotes.

Which vertebrate genes are without introns?

genes encoding histones and interferons only.

Why do humans have so many introns?

introns make it possible to shuffle exons... maybe why introsn arose in the first place. It allows humans to have many proteins but only a couple of genes (due to shifting). **used for alternative splicing/exon shuffling... counter to "one gene, one enzyme" rule.

Key differences b/w transcripts produced in prokaryotes and eukaryotes.

The transcript produce by prokaryotes is NOT processed while the one made by eukaryotes must be processed. In prokarotes, if genes that are needed at the same time are located adjacent to each other, they will both be transcribed as a single mRNA <---called polycistronic mRNA. In eukaryotes, only a single gene is compied and includes introns.

Polycistronic mRNA

transcript that has several genes or cistrons. Within a polycistronic mRNA, each proein coding region is preceded by its own ribosome-binding site and AUG initation code which allows translation to occur in multiple places. **genes coded in a polycistronic mRNA are usually part of an operon (the resultant proteins work together as part of a metabolic pathway)

Transcription: Eukaryotes vs. Prokaryotes.

Transcription in eukaryotes is done in the nucleus such that the mRNA must be processed before it can leave the nucleus and be translated in the cytosol by ribosomes. In contrast, for prokaryotes, translation begins before RNA poly even finish transcribing. Eukaryotic transcripts come in pieces and thus must be proccessed while prokaryotes don't.

What type of bond is formed between the anticodom and the codon?

Hydrogen bonds

Translation: steps

initiaon --> elongation --> termination. Where mRNA is translated into AA sequency by tRNA at a ribosome. mRNA is translated 3 nucleotides (codon) at a time.

How many AA does the genetic code specify?

20

What is the purpose of mRNA?

specifies polypeptide chain

What is the purpose of ribosome?

made up of a large and samll subunit that come together during translation; sandwiches the mRNA.

What is the purpose of tRNA?

tRNA are molecules that transport requred amino acids to ribosome for assembly.

What is the purpose of aminoacyl-tRNA synthetases

they catalyze attachment of amino acids to their particular tRNAs. What actually attaches the AA to the tRNA....once this occurs, the tRNA is referred to as a charged tRNA or an aminoacylated tRNA.

Prokaryotic vs. Eukaryotic mRNA

Prokaryotic mRNA -- no processing, translation begins before transcription ends (in cytoplasm), may contain info for more than one protein (polycistronic mRNA). For eukaryotes --> process, transcription and translation are spatially and temporally seperated, and only contains code for one protein.

Location of transc./translation: prokaryotes vs. eukaryotes

Prokarytes = everything happens in the cytoplasm and concurrently. Eukaryotes = transcription occurs in nucleus and must be finished completely before translation can occur which happens in the cytoplasm.

Where can ribosomes initiate transcription: prokaryotes vs. eukaryotes

For prokayotic ribosomes, they can initiate translation anywhere within an mRNA as long as it can find AUG (methionine; the start codon) downstream of the Shine-Dalgarno seq (AGGAGG). For eukaryotes, the ribosome can only iniated translation at the 5'end and thus can only produce 1 polypeptide.

What is a cistron

DNA sequence that encodes a single polypeptide chain

What is an operon

genes that share a promotor are coexpressed. **only have a few eukaryotic operons.

Ribosome: general

macromolecule made up of rRNA, proteins for scaffolding, and two subunits (large and small; small is where the mRNA binds to). Contains 3 ribosomal sites: A (aminoacyl) site, P (polypeptide) site, and E (exit)site.

What is the A site for on a ribosmoe?

accepts incoming tRNA holding AA

What is the P site for?

holds growing AA chain.

Each ribosomal site is made up of how many nucleotides?

3!!! Because they each accept a codon which is 3 nucleotides long.

Where are proteins for export made?

ribosomes bound to RER.

What type of ribosomes do prokaryotes have?

free ribosomes

How does the anticodon on the tRNA pair with the mRNA codon?

antiparallel and complementary

What holds the AA on an activated tRNA?

the 3'acceptor stem holds the amino acid; attachment of the AA was done via the aminoacyl-tRNA synthetases.

How many different aminoacyl-tRNA synthetases do we have for translation?

one for each different tRNA.

Translations: initiation

mRNA and initiation factors bind to small subunit of ribosome; in prokaryotes, the ribosome binds to the Shine-Dalgarno sequence and in eukaryotes, ribosomes bind to 5' cap. In both cases, the places where the ribosome binds are in the 5'UTR region of the mRNA. Next, the initiation complex (which already includes a tRNA-Met (initiator tRNA) scans in the 3' direction for a start codon. Once found, tRNA-Met (iniator tRNA) binds to AUG codon at P site...in which all subsequent binding are at the A site. Lastly, the Large ribosomal subunit sits on top of everything like a hamburger and binds to the initiation complex. **Basically, it goes: initiation factors --> small subunit/tRNA-met --> scanning --> large subunit.

Translation: elongation

the ribosome moves down the mRNA codon one at a time. New aminoacylated tRNA with the matching anticodon are brought into the A site (aa specified by codon in A site is brought in with the help of elongation factors). A peptide bond is formed between the AA in the A and P site with the help of peptidyl transferase. Covalent bond b/w tRNA and AA in P site is broken via hydrolysis leaving an uncharged tRNA in the P site and a dipeptide in the A site. Everything is shifted down one spot in the 3 ribosomal site such that the dipeptide in the A site is now in the P site and a new codon is in the A site ready for its anticodon tRNA... the process keeps repeating.

Translation: termination

Ribosome encounters a stop codon (UAG, UAA, UGA). Release factor binds to ribosome in response to stop codon so that no tRNA can bind to that site which causes the release of the newly formed polypeptide and the remaining bound tRNA. Ribosomal complex dissociates.

Peptidyl transferase

enzyme that catalyzes peptide bond formation b/w AA. Contained in the large ribosomal subunit.

Key differences b/w translation in eukaryotes and prokaryotes

For prokaryotes, translation can result in many gene products/proteins that are often part of a cistron. In eukaryotes, the initiation complex scans from the 5' cap to locate the first AUG; in prokaryotes, the initiation complex forms at ANY ribosome binding site in the mRNA..

The genetic code refers to what?

the mRNA sequence NOT the DNA sequence.

Characteristics of the genetic code

Code is written in linear form using mRNA bases. Made up of codons (3 nucleotides). It is unambiguous (each codon only codes for one AA), redundant (1 AA has more than 1 codon), degenerate (3rd and sometimes 2nd position often don't matter in determining the identity of AA). Code has stop and start codons. Code is non overlapping/fixed reading frame (once translation occurs, each base is read in order with no overlap). Code is nearly universal and is used by all living things (excpet for mitochondral genomes).

Why is it that while we have 61 codons, we only have approx. 40 tRNAs?

Due to Wobble hypothesis (F.Crick) which states that because the 3rd base in the mRNA codon is less constrainted than the first 2 bases, this allows a single tRNA to bind to more than 1 mRNA codon (hence the "wobbling"). For example, if the 5' end of the anticodon has a U, it can bind to either a A or G in the 3' end of the codon. If the 5' end of the anticodon contains I (inoside -- nucleoside found mainly in tRNA), it can bind to U, C, or A at the 3' end of the codon. however, rules vary among organisms.

What is the start codon?

AUG. METHIONINE!!

What happens to methionine after translation occurs?

it gets removed.

What does AUG in the middle of an eukaryotic sequence read as?

methio9.

What is the first AA inserted for prokaryotes?

a modified form of methionine (N-formylmethio9 aka f-met).

Do the stop codons pair with tRNA/code for any AA?

no

Universality of genetic code

nearly universal (including viruses, bacteria) except for mitochondria, mycoplasma, and some protozoans. The variation in the code usually is a result of a change in the recognition of the wobble position. Ex. UGA is a stop codon in the nuclear genetic code by is typtophan in the mitocondrial code.

When can control of eukaryotic gene expression occur?

during transcription via DNA binding proteins, after transcription via RNA binding proteins, and then post translation via folding or something. ***also the promotor strength is a good way to control gene expression.

What is fragile S Mental retardation protein (FMRP) an example of?

The importance of proteins that help with regulation of translation. Fragile X syndrome (resulting in mental retardation) is a result of mutations in the gene enconding FMRP (from trasncriptional silencing or a single AA change) such that translation of FMRP-associated mRNAs doesn't occur.

Protein structure

Shape is important for function. Shape is determined by AA sequence. Function can sometimes be predicted from shape.

What ultimately determines the shape and function of proteins?

nucleotide sequences of gene ultimately but we can say it is also due to the AA sequence due to colinearity (correspondance) b/w DNA nucleotide sequence and AA sequence of protein.

what are domains?

active sites of proteins/enzymes. Regions that interact with substrates.

What is the direction of polypeptide synthesis/ translation?

5' to 3'. From start to stop codon. Amino terminus of polypeptide is synthesized first which is near the 5' end of the mRNA whiel the carboxyl terminus is syntheiszed last (near 3' end of mRNA).

How are proteins folded?

in many different ways. 1-4 structures. Primary structure is the AA sequence. Secondary structure is the alpha or beta pleated sheets as resulting from the interactions between R groups. Tertiary structure is when you get disulfide bonds, hydrophobic interactions, van der Waal forces, etc. Quaternary structure is 2+ polypeptides coming togehter.

What do chaperone proteins do?

They help in protein folding for more complex, large multi-domain proteins and especially when proteins are under stress conditions (like when hydrophobic regions get exposed and form inactive aggregates... chaperons bind to the hydrophobic regions and prevent aggregation/allow time for proper folding to occur).

T or F: Most polypeptide chains fold correctly when released from the ribosome.

T. mostly. When they aren't, chaperones step in.

How many codons do we have? How many specify an AA?

we have 64 codons (mRNA). 61 specify an AA.

How many tRNAs do we have?

40 anticodons/tRNA even though we have 61 codons... possible due to Wobble hypothesis.

What is the effect of mutations in coding regions?

can alter gene function by altering amount or structure of protein product.

Types of mutations

substitution/missense, premature stop/nonsense, and frame shift.

Substitution/missense mutations

occurs when you have a single base pair change in the DNA leading to the addition/substiution of a different AA... "missense"

Premature stop/nonsense mutation

when a change in the DNA causes a stop codon to form prematurely.

Frameshift mutation

insertion or deletion of one or two nucleotides altering the reading frame because it alters all downstream codons.

What is sickle-cell anemia an example of?

molecular basis of sickle-cell anemia is mutation in the gene encoding B-globin. The mutation caused a single nucleotide base change: GAG to GUG... **due to missense mutation.

What causes mutations?

errors during DNA synthesis, crossing over, or intron removal.

In high temp, what types of proteins are needed to help in protein folding?

heat shock proteins

Where the mutation occurs dictates the effects. How so?

Mutation in promotor or exon regions are worse and result in null products (either doesn't get made or are not functional.) If mutation occurs in intron, nothing happens. Everything is ok and mutation is silent. If muation occurs at a border b/w intron and exon, you get a "leaky" mutation meaning some phenotypic effect.

When would you get a null result for a mutation in a noncoding/intron region?

When the mutation occurs at a spliceosome sequence.

Mutations: general

any HERITABLE change in the genetic material within cell ines or families. MUTATIONS ARE THE ULTIMATE SOURCE OF GENETIC VARIATION!!!! Mutations are good or bad. Mutations are spontaneous, statstically random events --> timing and cellular locations are unpredictable. HOWEVER, nonramdo with respect to location in the genome.

T or F: each gene has a characteristic mutation rate.

T.

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